Silk protein fragment compositions and articles manufactured therefrom

ABSTRACT

A composition is disclosed that includes pure silk fibroin-based protein fragments that are substantially devoid of sericin, wherein the composition has an average weight average molecular weight ranging from about 17 kDa to about 38 kDa, wherein the composition has a polydispersity of between about 1.5 and about 3.0, wherein the composition is substantially homogeneous, wherein the composition between 0 ppm to about 500 ppm of inorganic residuals, and wherein the composition includes between 0 ppm to about 500 ppm of organic residuals.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Application No. 61/884,820, filed Sep. 30, 2013, U.S.Provisional Application No. 62/000,928, filed May 20, 2014, and U.S.Provisional Application No. 62/036,450, filed Aug. 12, 2014. Thecontents of each of these applications are incorporated herein byreference in their entirety.

BACKGROUND

Silk is a natural polymer produced by a variety of insects and spiders.Silk comprises a filament core protein, silk fibroin, and a glue-likecoating consisting of a non-filamentous protein, sericin. Silk has beenhistorically studied for use in the medical field. Silk has been welldescribed in its natural fibrous form and is being studied further forpotentially useful secondary forms such as silk gels, sponges, serums,films, powders and composites. Many of these secondary forms can only becreated after processing the silk fibers into an aqueous silk solution.

Silk solutions have been generated using a variety of methods with thefinal solutions having a range of characteristics and varying levels ofpurity. Silk solutions have not only been used in medical applications,but have also expanded to other areas such as cosmetics and electronics.

SUMMARY

Silk protein fragment compositions and articles manufactured therefromare disclosed herein. Silk protein fragment compositions are furtherprocessed to remove water to varying levels resulting in a range ofarticles from lyophilized powder to aqueous gels. In an embodiment, anarticle of the present disclosure is a silk film. In an embodiment, asilk film of the present disclosure can be used to address fine linesand wrinkles of the skin, for example fine lines and wrinkles around themouth and nose. In an embodiment, a silk film of the present disclosurecan be used to address dark spots on the skin. In an embodiment, a silkfilm of the present disclosure is used for reducing puffy eyes. In anembodiment, an article of the present disclosure is a silk gel. In anembodiment, a silk gel of the present disclosure can be used as afirming eye gel. In an embodiment, a silk gel of the present disclosurecan replenish moisture and increase cell renewal while restoringradiance. In an embodiment, a silk gel of the present disclosure is asoothing gel. In an embodiment, a silk gel of the present disclosure isused for reducing puffy eyes. In an embodiment, a silk gel of thepresent disclosure is used for reducing dark circles around the eyes. Inan embodiment, an article of the present disclosure is a silk serum. Inan embodiment, a silk serum of the present disclosure can be used as ahydrating serum to restore hydration to the skin. In an embodiment, asilk serum of the present disclosure can be used to treat redness, acneand hyperpigmentation of the skin. In an embodiment, an article of thepresent disclosure is a silk chemical peel that damages the skin in acontrolled manner. In an embodiment, a silk chemical peel of the presentdisclosure, when applied to the skin, results in healthy vibrant skin.In an embodiment, a silk chemical peel of the present disclosure, whenapplied to the skin, results in a reduction in fine lines. In anembodiment, a silk chemical peel of the present disclosure, when appliedto the skin, results in firming of the skin. In an embodiment, anarticle of the present disclosure is a silk sunscreen gel.

According to aspects illustrated herein, methods for preparing aqueoussolutions of pure silk fibroin-based protein fragments are disclosed. Inan embodiment, at least one pure silk fibroin-based protein fragment(SPF) mixture solution having a specific average weight averagemolecular weight (MW) range and polydispersity is created. In anembodiment, at least SPF mixture solution having a MW range betweenabout 6 kDa and 16 kDa and a polydispersity range between about 1.5 andabout 3.0 is created. In an embodiment, at least one SPF mixturesolution having a MW between about 17 kDa and 38 kDa and apolydispersity range between about 1.5 and about 3.0 is created. In anembodiment, at least one SPF mixture solution having a MW range betweenabout 39 kDa and 80 kDa and a polydispersity range between about 1.5 andabout 3.0 is created.

According to aspects illustrated herein, there is disclosed acomposition that includes pure silk fibroin-based protein fragments thatare substantially devoid of sericin, wherein the composition has anaverage weight average molecular weight ranging from about 6 kDa toabout 16 kDa, wherein the composition has a polydispersity of betweenabout 1.5 and about 3.0, wherein the composition is substantiallyhomogenous, wherein the composition includes between 0 ppm and about 500ppm of inorganic residuals, and wherein the composition includes between0 ppm and about 500 ppm of organic residuals. In an embodiment, the puresilk fibroin-based protein fragments have between about 10 ppm and about300 ppm of lithium bromide residuals and between about 10 ppm and about100 ppm of sodium carbonate residuals. In an embodiment, the lithiumbromide residuals are measurable using a high-performance liquidchromatography lithium bromide assay, and the sodium carbonate residualsare measurable using a high-performance liquid chromatography sodiumcarbonate assay. In an embodiment, the composition further includes lessthan 10% water. In an embodiment, the composition is in the form of alyophilized structure, such as a lyophilized powder. In an embodiment,the composition is in the form of a solution. In an embodiment, thecomposition includes from about 0.1 wt % to about 30.0 wt % pure silkfibroin-based protein fragments. The pure silk fibroin-based proteinfragments are stable in the solution for at least 30 days. In anembodiment, the term “stable” refers to the absence of spontaneous orgradual gelation, with no visible change in the color or turbidity ofthe solution. In an embodiment, the term “stable” refers to noaggregation of fragments and therefore no increase in molecular weightover time. In an embodiment, the composition is in the form of anaqueous solution. In an embodiment, the composition is in the form of anorganic solution. The composition may be provided in a sealed container.In some embodiments, the composition further includes one or moremolecules selected from the group consisting of therapeutic agents,growth factors, antioxidants, proteins, vitamins, carbohydrates,polymers, nucleic acids, salts, acids, bases, biomolecules, glycosaminoglycans, polysaccharides, extracellular matrix molecules, metals, metalion, metal oxide, synthetic molecules, polyanhydrides, cells, fattyacids, fragrance, minerals, plants, plant extracts, preservatives andessential oils. In an embodiment, the added molecule or molecules arestable (i.e., retain activity over time) within the composition and canbe released at a desired rate. In an embodiment, the one or moremolecules is vitamin C or a derivative thereof. In an embodiment, thecomposition further includes an alpha hydroxy acid selected from thegroup consisting of glycolic acid, lactic acid, tartaric acid and citricacid. In an embodiment, the composition further includes hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0%. In anembodiment, the composition further includes at least one of zinc oxideor titanium dioxide. In an embodiment, the pure silk fibroin-basedprotein fragments in the composition are hypoallergenic. In anembodiment, the pure silk fibroin-based protein fragments arebiocompatible, non-sensitizing, and non-immunogenic. In an embodiment,the pure silk fibroin-based protein fragments are bioresorbable orbiodegradable following implantation or application.

According to aspects illustrated herein, there is disclosed acomposition that includes pure silk fibroin-based protein fragments thatare substantially devoid of sericin, wherein the composition has anaverage weight average molecular weight ranging from about 17 kDa toabout 38 kDa, wherein the composition has a polydispersity of betweenabout 1.5 and about 3.0, wherein the composition is substantiallyhomogenous, wherein the composition includes between 0 ppm and about 500ppm of inorganic residuals, and wherein the composition includes between0 ppm and about 500 ppm of organic residuals. In an embodiment, the puresilk fibroin-based protein fragments have between about 10 ppm and about300 ppm of lithium bromide residuals and between about 10 ppm and about100 ppm of sodium carbonate residuals. In an embodiment, the lithiumbromide residuals are measurable using a high-performance liquidchromatography lithium bromide assay, and the sodium carbonate residualsare measurable using a high-performance liquid chromatography sodiumcarbonate assay. In an embodiment, the composition further includes lessthan 10% water. In an embodiment, the composition is in the form of alyophilized structure, such as a lyophilized powder. In an embodiment,the composition is in the form of a solution. In an embodiment, thecomposition includes from about 0.1 wt % to about 30.0 wt % pure silkfibroin-based protein fragments. The pure silk fibroin-based proteinfragments are stable in the solution for at least 30 days. In anembodiment, the term “stable” refers to the absence of spontaneous orgradual gelation, with no visible change in the color or turbidity ofthe solution. In an embodiment, the term “stable” refers to noaggregation of fragments and therefore no increase in molecular weightover time. In an embodiment, the composition is in the form of anaqueous solution. In an embodiment, the composition is in the form of anorganic solution. The composition may be provided in a sealed container.In some embodiments, the composition further includes one or moremolecules selected from the group consisting of therapeutic agents,growth factors, antioxidants, proteins, vitamins, carbohydrates,polymers, nucleic acids, salts, acids, bases, biomolecules, glycosaminoglycans, polysaccharides, extracellular matrix molecules, metals, metalion, metal oxide, synthetic molecules, polyanhydrides, cells, fattyacids, fragrance, minerals, plants, plant extracts, preservatives andessential oils. In an embodiment, the added molecule or molecules arestable (i.e., retain activity over time) within the composition and canbe released at a desired rate. In an embodiment, the one or moremolecules is vitamin C or a derivative thereof. In an embodiment, thecomposition further includes an alpha hydroxy acid selected from thegroup consisting of glycolic acid, lactic acid, tartaric acid and citricacid. In an embodiment, the composition further includes hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0%. In anembodiment, the composition further includes at least one of zinc oxideor titanium dioxide. In an embodiment, the pure silk fibroin-basedprotein fragments in the composition are hypoallergenic. In anembodiment, the pure silk fibroin-based protein fragments arebiocompatible, non-sensitizing, and non-immunogenic. In an embodiment,the pure silk fibroin-based protein fragments are bioresorbable orbiodegradable following implantation or application.

According to aspects illustrated herein, there is disclosed acomposition that includes pure silk fibroin-based protein fragments thatare substantially devoid of sericin, wherein the composition has anaverage weight average molecular weight ranging from about 39 kDa toabout 80 kDa, wherein the composition has a polydispersity of betweenabout 1.5 and about 3.0, wherein the composition is substantiallyhomogenous, wherein the composition includes between 0 ppm and about 500ppm of inorganic residuals, and wherein the composition includes between0 ppm and about 500 ppm of organic residuals. In an embodiment, the puresilk fibroin-based protein fragments have between about 10 ppm and about300 ppm of lithium bromide residuals and between about 10 ppm and about100 ppm of sodium carbonate residuals. In an embodiment, the lithiumbromide residuals are measurable using a high-performance liquidchromatography lithium bromide assay, and the sodium carbonate residualsare measurable using a high-performance liquid chromatography sodiumcarbonate assay. In an embodiment, the composition further includes lessthan 10% water. In an embodiment, the composition is in the form of alyophilized structure, such as a lyophilized powder. In an embodiment,the composition is in the form of a solution. In an embodiment, thecomposition includes from about 0.1 wt % to about 30.0 wt % pure silkfibroin-based protein fragments. The pure silk fibroin-based proteinfragments are stable in the solution for at least 30 days. In anembodiment, the term “stable” refers to the absence of spontaneous orgradual gelation, with no visible change in the color or turbidity ofthe solution. In an embodiment, the term “stable” refers to noaggregation of fragments and therefore no increase in molecular weightover time. In an embodiment, the composition is in the form of anaqueous solution. In an embodiment, the composition is in the form of anorganic solution. The composition may be provided in a sealed container.In some embodiments, the composition further includes one or moremolecules selected from the group consisting of therapeutic agents,growth factors, antioxidants, proteins, vitamins, carbohydrates,polymers, nucleic acids, salts, acids, bases, biomolecules, glycosaminoglycans, polysaccharides, extracellular matrix molecules, metals, metalion, metal oxide, synthetic molecules, polyanhydrides, cells, fattyacids, fragrance, minerals, plants, plant extracts, preservatives andessential oils. In an embodiment, the added molecule or molecules arestable (i.e., retain activity over time) within the composition and canbe released at a desired rate. In an embodiment, the one or moremolecules is vitamin C or a derivative thereof. In an embodiment, thecomposition further includes an alpha hydroxy acid selected from thegroup consisting of glycolic acid, lactic acid, tartaric acid and citricacid. In an embodiment, the composition further includes hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0%. In anembodiment, the composition further includes at least one of zinc oxideor titanium dioxide. In an embodiment, the pure silk fibroin-basedprotein fragments in the composition are hypoallergenic. In anembodiment, the pure silk fibroin-based protein fragments arebiocompatible, non-sensitizing, and non-immunogenic. In an embodiment,the pure silk fibroin-based protein fragments are bioresorbable orbiodegradable following implantation or application.

According to aspects illustrated herein, there is disclosed a film thatincludes pure silk fibroin-based protein fragments substantially devoidof sericin and comprising: an average weight average molecular weightranging from about 17 kDa to about 38 kDa; and a polydispersity ofbetween about 1.5 and about 3.0, wherein the film has a water contentranging from about 2.0 wt. % to about 20.0 wt. %, wherein the filmincludes between 0 ppm and 500 ppm of inorganic residuals, wherein thefilm includes between 0 ppm and 500 ppm of organic residuals, andwherein the film is sufficiently flexible to conform to anatomicaltopographies. In an embodiment, the film includes between about 1.0% andabout 50.0% crystalline protein domains and being soluble when submersedin water at room temperature. In an embodiment, the film includes fromabout 30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based proteinfragments. In an embodiment, the film has a pH from about 1.0 to about7.0. In an embodiment, the film further includes from about 0.5 wt. % toabout 2.5 wt. % of caffeine. In an embodiment, the film further includesfrom about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivativethereof. In an embodiment, the vitamin C or a derivative thereof remainsstable within the film for a period of from about 5 days to about 5years. In an embodiment, the vitamin C or a derivative thereof is stablewithin the film so as to result in release of the vitamin C in abiologically active form. In an embodiment, the film further includesone or more molecules selected from the group consisting of therapeuticagents, growth factors, antioxidants, proteins, carbohydrates, polymers,nucleic acids, salts, acids, bases, biomolecules, glycosamino glycans,polysaccharides, extracellular matrix molecules, metals, metal ion,metal oxide, synthetic molecules, polyanhydrides, cells, fatty acids,fragrance, minerals, plants, plant extracts, preservatives and essentialoils. In an embodiment, the film further includes an alpha hydroxy acidselected from the group consisting of glycolic acid, lactic acid,tartaric acid and citric acid. In an embodiment, the film furtherincludes hyaluronic acid or its salt form at a concentration rangingfrom about 0.5 wt. % to about 10.0 wt. %. In an embodiment, the filmfurther includes at least one of zinc oxide or titanium dioxide. In anembodiment, the film is packaged in a foil based package that is airtight and light proof. In an embodiment, the film is sufficientlydesigned for topical application. In an embodiment, the topicalapplication is for cosmetic use. In an embodiment, the topicalapplication is for wound dressing. In an embodiment, the film issufficiently designed for administration within a body. In anembodiment, the pure silk fibroin-based protein fragments arehypoallergenic. In an embodiment, a method of reducing fine lines andwrinkles includes applying a film of the present disclosure daily tohuman skin for a period of at least one week and observing a reductionin fine lines and wrinkles on the human skin.

According to aspects illustrated herein, there is disclosed a gel thatincludes pure silk fibroin-based protein fragments substantially devoidof sericin and comprising: an average weight average molecular weightranging from about 17 kDa to about 38 kDa; and a polydispersity ofbetween about 1.5 and about 3.0; and water from about 20 wt. % to about99.9 wt. %, wherein the gel includes between 0 ppm and 500 ppm ofinorganic residuals, and wherein the gel includes between 0 ppm and 500ppm of organic residuals. In an embodiment, the gel includes betweenabout 1.0% and about 50.0% crystalline protein domains. In anembodiment, the gel includes from about 0.1 wt. % to about 6.0 wt. % ofpure silk fibroin-based protein fragments. In an embodiment, the gel hasa pH from about 1.0 to about 7.0. In an embodiment, the gel furtherincludes from about 0.5 wt. % to about 20.0 wt. % of vitamin C or aderivative thereof. In an embodiment, the vitamin C or a derivativethereof remains stable within the gel for a period of from about 5 daysto about 5 years. In an embodiment, the vitamin C or a derivativethereof is stable within the gel so as to result in release of thevitamin C in a biologically active form. In an embodiment, the gelfurther includes an additive selected from the group consisting ofvitamin E, rosemary oil, rose oil, lemon juice, lemon grass oil andcaffeine. In an embodiment, the gel is packaged in an airtightcontainer. In an embodiment, the pure silk fibroin-based proteinfragments are hypoallergenic. In an embodiment, the gel has less than 10colony forming units per milliliter. In an embodiment, a method ofsmoothing and rejuvenating human skin includes applying a gel of thepresent disclosure daily to human skin for a period of at least one weekand observing an improvement in skin texture.

According to aspects illustrated herein, there is disclosed a serum thatincludes pure silk fibroin-based protein fragments substantially devoidof sericin and comprising: an average weight average molecular weightranging from about 17 kDa to about 38 kDa; and a polydispersity ofbetween about 1.5 and about 3.0; and hyaluronic acid or its salt formfrom about 0.5% to aboutl0.0%, wherein the serum includes between 0 ppmand 500 ppm of inorganic residuals, and wherein the serum includesbetween 0 ppm and 500 ppm of organic residuals. In an embodiment, theserum includes between about 1.0% and about 50.0% crystalline proteindomains. In an embodiment, the serum includes from about 0.1 wt. % toabout 6.0 wt. % of pure silk fibroin-based protein fragments. In anembodiment, the serum has a pH from about 1.0 to about 7.0. In anembodiment, the serum further includes an additive selected from thegroup consisting of vitamin E, rosemary oil, rose oil, lemon juice,lemon grass oil, vanilla, geranium, and green tea. In an embodiment, theserum further includes from about 0.5 wt. % to about 30.0 wt. % ofvitamin C or a derivative thereof. In an embodiment, the vitamin C or aderivative thereof remains stable within the serum for a period of fromabout 5 days to about 5 years. In an embodiment, the vitamin C or aderivative thereof is stable within the serum so as to result in releaseof the vitamin C in a biologically active form. In an embodiment, theserum is packaged in an airtight container. In an embodiment, the puresilk fibroin-based protein fragments are hypoallergenic. In anembodiment, a method of moisturizing human skin includes applying dailya serum of the present disclosure to human skin for a period of at leastone week and observing an improvement in skin hydration.

According to aspects illustrated herein, there is disclosed a skin peelcomposition that includes pure silk fibroin-based protein fragments thatare substantially devoid of sericin, the fragments having an averageweight average molecular weight ranging from about 17 kDa to about 38kDa and a polydispersity of between about 1.5 and about 3.0 incombination with at least one skin exfoliating agent. In an embodiment,the skin peel composition includes at least one skin exfoliating agentselected from the group consisting of glycolic acid and lactic acid. Inan embodiment, the skin peel composition includes between about 1.0% andabout 50.0% crystalline protein domains. In an embodiment, the skin peelcomposition has a pH from about 1.0 to about 6.0. In an embodiment, thepure silk fibroin-based protein fragments are hypoallergenic.

According to aspects illustrated herein, there is disclosed a method forpreparing an aqueous solution of pure silk fibroin-based proteinfragments having an average weight average molecular weight ranging fromabout 6 kDa to about 16 kDa, the method including the steps of:degumming a silk source by adding the silk source to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 60° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in an ovenhaving a temperature of about 140° C. for a period of at least 1 hour;removing the lithium bromide from the silk fibroin extract; andproducing an aqueous solution of silk protein fragments, the aqueoussolution comprising: fragments having an average weight averagemolecular weight ranging from about 6 kDa to about 16 kDa, and whereinthe aqueous solution of pure silk fibroin-based protein fragmentscomprises a polydispersity of between about 1.5 and about 3.0. In anembodiment, the method includes the step of drying the silk fibroinextract prior to the dissolving step. In an embodiment, the amount oflithium bromide residuals in the aqueous solution can be measured usinga high-performance liquid chromatography lithium bromide assay. In anembodiment, the amount of sodium carbonate residuals in the aqueoussolution can be measured using a high-performance liquid chromatographysodium carbonate assay. In an embodiment, the method includes the stepof adding a therapeutic agent to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the method includesthe step of adding a molecule selected from one of an antioxidant or anenzyme to the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, the method includes the step of adding avitamin to the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, the vitamin is selected from one of vitaminC or a derivative thereof. In an embodiment, the method further includesthe step of adding an alpha hydroxy acid to the aqueous solution of puresilk fibroin-based protein fragments. In an embodiment, the alphahydroxy acid is selected from the group consisting of glycolic acid,lactic acid, tartaric acid and citric acid. In an embodiment, the methodfurther includes the step of adding hyaluronic acid at a concentrationof about 0.5% to about 10.0% to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the method furtherincludes the step of adding at least one of zinc oxide or titaniumdioxide to the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, the method further includes the step oflyophilizing the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, a cosmetic film is fabricated from theaqueous solution of silk protein fragments. In an embodiment, a cosmeticgel is fabricated from the aqueous solution of silk protein fragments.

According to aspects illustrated herein, there is disclosed a method forpreparing an aqueous solution of pure silk fibroin-based proteinfragments having an average weight average molecular weight ranging fromabout 17 kDa to about 38 kDa, the method including the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of between about 30 minutes to about 60minutes so as to result in degumming; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 80° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in a dry ovenhaving a temperature in the range between about 60° C. to about 100° C.for a period of at least 1 hour; removing the lithium bromide from thesilk fibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, wherein the aqueous solutionof silk protein fragments comprises sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, wherein the aqueous solution ofpure silk fibroin-based protein fragments comprises fragments having anaverage weight average molecular weight ranging from about 17 kDa toabout 38 kDa, and wherein the aqueous solution of pure silkfibroin-based protein fragments comprises a polydispersity of betweenabout 1.5 and about 3.0. In an embodiment, the method includes the stepof drying the silk fibroin extract prior to the dissolving step. In anembodiment, the amount of lithium bromide residuals in the aqueoussolution can be measured using a high-performance liquid chromatographylithium bromide assay. In an embodiment, the amount of sodium carbonateresiduals in the aqueous solution can be measured using ahigh-performance liquid chromatography sodium carbonate assay. In anembodiment, the method includes the step of adding a therapeutic agentto the aqueous solution of pure silk fibroin-based protein fragments. Inan embodiment, the method includes the step of adding a moleculeselected from one of an antioxidant or an enzyme to the aqueous solutionof pure silk fibroin-based protein fragments. In an embodiment, themethod includes the step of adding a vitamin to the aqueous solution ofpure silk fibroin-based protein fragments. In an embodiment, the vitaminis selected from one of vitamin C or a derivative thereof. In anembodiment, the method further includes the step of adding an alphahydroxy acid to the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, the alpha hydroxy acid is selected from thegroup consisting of glycolic acid, lactic acid, tartaric acid and citricacid. In an embodiment, the method further includes the step of addinghyaluronic acid at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. In anembodiment, the method further includes the step of adding at least oneof zinc oxide or titanium dioxide to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the method furtherincludes the step of lyophilizing the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, a cosmetic film isfabricated from the aqueous solution of silk protein fragments. In anembodiment, a cosmetic gel is fabricated from the aqueous solution ofsilk protein fragments.

According to aspects illustrated herein, there is disclosed a method forpreparing an aqueous solution of pure silk fibroin-based proteinfragments having an average weight average molecular weight ranging fromabout 39 kDa to about 80 kDa, the method including the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of about 30 minutes so as to result indegumming; removing sericin from the solution to produce a silk fibroinextract comprising non-detectable levels of sericin; draining thesolution from the silk fibroin extract; dissolving the silk fibroinextract in a solution of lithium bromide having a starting temperatureupon placement of the silk fibroin extract in the lithium bromidesolution that ranges from about 80° C. to about 140° C.; maintaining thesolution of silk fibroin-lithium bromide in a dry oven having atemperature in the range between about 60° C. to about 100° C. for aperiod of at least 1 hour; removing the lithium bromide from the silkfibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, fragments having an averageweight average molecular weight ranging from about 40 kDa to about 65kDa, and wherein the aqueous solution of pure silk fibroin-based proteinfragments comprises a polydispersity of between about 1.5 and about 3.0.In an embodiment, the method includes the step of drying the silkfibroin extract prior to the dissolving step. In an embodiment, theamount of lithium bromide residuals in the aqueous solution can bemeasured using a high-performance liquid chromatography lithium bromideassay. In an embodiment, the amount of sodium carbonate residuals in theaqueous solution can be measured using a high-performance liquidchromatography sodium carbonate assay. In an embodiment, the methodincludes the step of adding a therapeutic agent to the aqueous solutionof pure silk fibroin-based protein fragments. In an embodiment, themethod includes the step of adding a molecule selected from one of anantioxidant or an enzyme to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the method includesthe step of adding a vitamin to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the vitamin isselected from one of vitamin C or a derivative thereof. In anembodiment, the method further includes the step of adding an alphahydroxy acid to the aqueous solution of pure silk fibroin-based proteinfragments. In an embodiment, the alpha hydroxy acid is selected from thegroup consisting of glycolic acid, lactic acid, tartaric acid and citricacid. In an embodiment, the method further includes the step of addinghyaluronic acid at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. In anembodiment, the method further includes the step of adding at least oneof zinc oxide or titanium dioxide to the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, the method furtherincludes the step of lyophilizing the aqueous solution of pure silkfibroin-based protein fragments. In an embodiment, a cosmetic film isfabricated from the aqueous solution of silk protein fragments. In anembodiment, a cosmetic gel is fabricated from the aqueous solution ofsilk protein fragments.

According to aspects illustrated herein, silk films manufactured fromSPF mixture solutions of the present disclosure are disclosed. In anembodiment, at least one molecule or therapeutic agent of interest isphysically entrapped into a SPF mixture solution of the presentdisclosure during processing into films. A silk film of the presentdisclosure can be used to release at least one molecule or therapeuticagent of interest.

According to aspects illustrated herein, a method is disclosed forproducing silk films having entrapped molecules or therapeutic agents.

According to aspects illustrated herein, a method is disclosed forproducing silk gels having entrapped molecules or therapeutic agentssuch as those listed in the following paragraph. In an embodiment, atleast one molecule or therapeutic agent of interest is physicallyentrapped into a SPF mixture solution of the present disclosure duringprocessing into aqueous gels. An aqueous silk gel of the presentdisclosure can be used to release at least one molecule or therapeuticagent of interest.

According to aspects illustrated herein, a SPF mixture solution of thepresent disclosure is used to fabricate a silk film or aqueous gel thatentraps molecules including, but not limited to, Selenium, Ubiquinonederivatives, Thiol-based antioxidants, Saccharide-containingantioxidants, Polyphenols, Botanical extracts, Caffeic acid, Apigenin,Pycnogenol, Resveratrol, Folic acid, Vitamin b12, Vitamin b6, Vitaminb3, Vitamin E, Vitamin C and derivatives thereof, Vitamin D, Vitamin A,Astaxathin, Lutein, Lycopene, Essential fatty acids (omegas 3 and 6),Iron, Zinc, magnesium, Flavonoids (soy, Curcumin, Silymarin,Pycnongeol), Growth factors, aloe, hyaluronic acid, extracellular matrixproteins, cells, nucleic acids, biomarkers, biological reagents, zincoxide, benzyol peroxide, retnoids, titanium, caffeine, green tea,allergens in a known dose (for sensitization treatment), essential oilsincluding, but not limited to, lemongrass or rosemary oil, andfragrances. A film of the present disclosure can adhere to the skin whenmoistened, allowing for easy application and targeted delivery to atreatment area with an ability to be wiped off with water. Moleculeloaded films, gels or serums can be used for drug delivery, medical andpersonal care, including anti-aging, wrinkle and fine line reduction andprevention, crows feet, frown lines and glabella line reduction; acnetreatment; UV protection; all types of wound care; topical, intradermaland sub-dermal and implantable medical and pharmaceutical applications;all types and conditions of inflammation, such as eczema or rosacea;creating an even skin tone, pigment reduction, hyperpigmentationtreatment, dark spots or age spots resulting from acne, pregnancy, birthcontrol, photodamage; reducing scar and stretch marks, and reducing acnescarring. By stabilizing molecules in a film or gel of the presentdisclosure, controlled release of the molecule in its active form isachieved. In an embodiment, a film or gel of the present disclosure candeliver its molecule in a time frame relevant to a consumer for dailyskin treatment(s). In an embodiment, the pure silk fibroin-based proteincomposition in an aqueous or organic solution may be used to spin fibersor fabrics for the medical or consumer markets.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed embodiments.

FIG. 1 is a flow chart showing various embodiments for producing puresilk fibroin-based protein fragments (SPFs) of the present disclosure.

FIG. 2 is a flow chart showing various parameters that can be modifiedduring the process of producing SPFs of the present disclosure duringthe extraction and the dissolution steps.

FIG. 3 is a photograph showing dry extracted silk fibroin.

FIG. 4 is a photograph showing an embodiment of a SPF in the form of asolution of the present disclosure.

FIGS. 5A-5D are photographs showing dissolved silk in room temperaturelithium bromide (LiBr) solutions dissolved in a 60° C. oven for 4 hours(sericin extraction temperature and time were varied).

FIGS. 6A-6D are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in a 60° C. oven for 6 hours (sericinextraction temperature and time were varied).

FIGS. 7A-7D are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in a 60° C. oven for 8 hours (sericinextraction temperature and time were varied).

FIGS. 8A-8D are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in a 60° C. oven for 12 hours (sericinextraction temperature and time were varied).

FIGS. 9A-9D are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in a 60° C. oven for 24 hours (sericinextraction temperature and time were varied).

FIGS. 10A-10C are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in a 60° C. oven for 168/192 hours (sericinextraction temperature and time were varied).

FIGS. 11A-11C are photographs showing dissolved silk in room temperatureLiBr solutions dissolved in 60° C. oven for 1, 4, and 6 hours, wheresericin extraction was completed at 100° C. for 60 min.

FIGS. 12A-12D are photographs showing dissolved silk in 60° C. LiBrsolutions dissolved in a 60° C. oven for 1 hour (sericin extractiontemperature and time were varied).

FIGS. 13A-13D are photographs showing dissolved silk in 60° C. LiBrsolutions dissolved in a 60° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 14A-14D are photographs showing dissolved silk in 60° C. LiBrsolutions dissolved in a 60° C. oven for 6 hours (sericin extractiontemperature and time were varied).

FIGS. 15A-15D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 60° C. oven for 1 hour (sericin extractiontemperature and time were varied).

FIGS. 16A-16D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 60° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 17A-17D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 60° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 18A-18D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 60° C. oven for 1 hour (sericin extractiontemperature and time were varied).

FIGS. 19A-19D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 60° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 20A-20D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 60° C. oven for 6 hours (sericin extractiontemperature and time were varied).

FIGS. 21A-21D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 60° C. oven for 1 hour(sericin extraction temperature and time were varied time).

FIGS. 22A-22D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 60° C. oven for 4 hours(sericin extraction temperature and time were varied).

FIGS. 23A-23D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 60° C. oven for 6 hours(sericin extraction temperature and time were varied).

FIGS. 24A-24D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 80° C. oven for 1 hour (sericin extractiontemperature and time were varied).

FIGS. 25A-25D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 80° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 26A-26D are photographs showing dissolved silk in 80° C. LiBrsolutions dissolved in a 80° C. oven for 6 hours (sericin extractiontemperature and time were varied).

FIGS. 27A-27D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 100° C. oven for 1 hour (sericin extractiontemperature and time were varied).

FIGS. 28A-28D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 100° C. oven for 4 hours (sericin extractiontemperature and time were varied).

FIGS. 29A-29D are photographs showing dissolved silk in 100° C. LiBrsolutions dissolved in a 100° C. oven for 6 hours (sericin extractiontemperature and time were varied).

FIGS. 30A-30D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 120° C. oven for 1 hour(sericin extraction temperature and time were varied).

FIGS. 31A-31D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 120° C. oven for 4 hours(sericin extraction temperature and time were varied).

FIG. 32A-32D are photographs showing dissolved silk in 140° C. (boilingpoint for LiBr) LiBr solutions dissolved in a 120° C. oven for 6 hours(sericin extraction temperature and time were varied).

FIG. 33 is a flow chart showing an embodiment for producing a silk filmof the present disclosure from a silk solution of the presentdisclosure.

FIG. 34 summarizes an embodiment of parameters for a silk film dryingstudy of the present disclosure.

FIG. 35 is a graph showing silk film drying times (under various airflow and temperature conditions).

FIGS. 36A and 36B show HPLC chromatograms from samples comprisingvitamin C. FIG. 36A shows peaks from (1) a chemically stabilized sampleof vitamin C at ambient conditions and (2) a sample of vitamin C takenafter 1 hour at ambient conditions without chemical stabilization toprevent oxidation, where degradation products are visible. FIG. 36Bshows peaks from two different embodiments of silk films of the presentdisclosure that were aged for at least 30 days at room temperature. Nodegradation products were visible.

FIGS. 37A-37D are photographs showing silk protein fragment-films of thepresent disclosure dried at room temperature for 48 hours with open airflow.

FIGS. 38A-38D are photographs showing silk protein fragment-films of thepresent disclosure dried at 40° C. in a convection oven for 8 hours withopen air flow.

FIGS. 39A-39D are photographs showing silk protein fragment-films of thepresent disclosure dried at 40° C. in a convection oven for 48 hourswith open air flow.

FIGS. 40A-40D are photographs showing silk protein fragment-films of thepresent disclosure dried at 40° C. in a convection oven for 48 hours inclosed dish.

FIGS. 41A-41D are photographs showing silk protein fragment-films of thepresent disclosure dried at 54° C. in a convection oven for 8 hours inopen dish.

FIGS. 42A-42D are photographs showing silk protein fragment-films of thepresent disclosure dried at 54° C. in a convection oven for 48 hours inopen dish.

FIGS. 43A-43D are photographs showing silk protein fragment-films of thepresent disclosure dried at 54° C. in a film dryer for 8 hours in opendish.

FIGS. 44A-44D are photographs showing silk protein fragment-films of thepresent disclosure dried at 54° C. in a film dryer for 48 hours in opendish.

FIGS. 45A-45D are photographs showing silk protein fragment-films of thepresent disclosure dried at room temperature in a convection oven for 48hours in open dish.

FIGS. 46A-46D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried atroom temperature for 48 hours with open air flow.

FIGS. 47A-47D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at40° C. in a convection oven for 8 hours with open air flow.

FIGS. 48A-48D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at40° C. in a convection oven for 48 hours with open air flow.

FIGS. 49A-49D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at40° C. in a convection oven for 48 hours in closed dish.

FIGS. 50A-50D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at54° C. in a convection oven for 8 hours in open dish.

FIGS. 51A-51D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at54° C. in a convection oven for 48 hours in open dish.

FIGS. 52A-52D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at54° C. in a film dryer for 8 hours in open dish.

FIGS. 53A-53D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried at54° C. in a film dryer for 48 hours in open dish.

FIGS. 54A-54D are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure dried atroom temperature in a convection oven for 48 hours in open dish.

FIG. 55 is a table summarizing the LiBr and Sodium Carbonate (Na₂CO₃)concentration in silk protein solutions of the present disclosure.

FIG. 56 is a table summarizing the Na₂CO₃ concentration in silk proteinfragment-films of the present disclosure.

FIG. 57 is a table summarizing the LiBr concentration in silk proteinfragment-films of the present disclosure.

FIG. 58 is a table summarizing the LiBr and Na₂CO₃ concentration in silkprotein solutions of the present disclosure.

FIG. 59 is a table summarizing the vitamin C concentration in silkprotein fragment-films of the present disclosure.

FIG. 60 is a table summarizing the stability of vitamin C in chemicallystabilized solutions.

FIG. 61 is a table summarizing the Molecular Weights of silk proteinsolutions of the present disclosure.

FIGS. 62A and 62B are graphs representing the effect of extractionvolume on % mass loss.

FIG. 63 is a table summarizing the Molecular Weights of silk dissolvedfrom different concentrations of LiBr and from different extraction anddissolution sizes.

FIG. 64 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 100° C. LiBr and 100° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 65 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, boiling LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 66 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 60° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 67 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 80° C. LiBr and 80° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 68 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 80° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 69 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 100° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 70 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 140° C. LiBr and 140° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 71 is a graph summarizing the effect of Extraction Temperature onMolecular Weight of silk processed under the conditions of 60 minuteExtraction Time, 100° C. LiBr and 100° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 72 is a graph summarizing the effect of LiBr Temperature onMolecular Weight of silk processed under the conditions of 60 minuteExtraction Time, 100° C. Extraction Temperature and 60° C. OvenDissolution (Oven/Dissolution Time was varied).

FIG. 73 is a graph summarizing the effect of LiBr Temperature onMolecular Weight of silk processed under the conditions of 30 minuteExtraction Time, 100° C. Extraction Temperature and 60° C. OvenDissolution (Oven/Dissolution Time was varied).

FIG. 74 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 30 minute Extraction Time, and 100°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 75 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 100°C. Lithium Bromide. (Oven/Dissolution Time was varied).

FIG. 76 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 140°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 77 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 30 minute Extraction Time, and 140°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 78 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 80° C.Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 79 is a graph summarizing the Molecular Weights of silk processedunder varying conditions including Extraction Time, ExtractionTemperature, Lithium Bromide (LiBr) Temperature, Oven Temperature forDissolution, Oven Time for Dissolution.

FIG. 80 is a graph summarizing the Molecular Weights of silk processedunder conditions in which Oven/Dissolution Temperature is equal to LiBrTemperature.

FIG. 81 is a graph representing the % Activity of Vitamin C in PureProC™Gel.

FIGS. 82A-82C are photographs showing the effect of film drying on filmcolor and physical integrity after storage (most dry (FIG. 82A), leastdry (FIG. 82C)).

FIGS. 83A and 83B are photographs of a laser cut silk film.

FIG. 84 is a graph summarizing the quantity of vitamin C in a daily dose(i.e., the average amount of product used to cover a 25 cm² area ofskin) of PureProC™ and competitor products over a 30 day period.

FIG. 85 is a graph summarizing the ease of use of PureProC™ collected ina user experience.

FIG. 86 is a summary of the initial benefits of PureProC™ observed byusers and support of consumer knowledge.

FIG. 87 is a graph summarizing where trial participants used PureProC™Smoothing Gel.

FIG. 88 is a summary of the benefits to the skin after using PureProC™Smoothing Gel: Lemongrass by trial participants.

FIGS. 89A-89B are tables summarizing the effect of vitamin C with orwithout a vitamin C derivative on gelation.

FIG. 90 is a table summarizing the effect of vitamin C and vitamin Cderivatives on the formation of silk films of the present disclosure.

FIGS. 91A-91B are tables summarizing the effect of vitamin C andcaffeine on the formation of silk films of the present disclosure.

FIG. 92 is a table summarizing an embodiment of a caffeine gel of thepresent disclosure.

FIG. 93 is a table summarizing embodiments of preservative gels of thepresent disclosure.

FIGS. 94A-94C are tables summarizing embodiments of cosmetic serums ofthe present disclosure with varying additives and concentrations ofcomponents suitable for protection against ultraviolet radiation (UV).

FIGS. 95A-95C are tables summarizing embodiments of high concentrationvitamin C gels of the present disclosure.

FIG. 96 is a table summarizing the results of various gels of thepresent disclosure to evaluate the possible microbial contamination inthree different states of their use (intact, in-use, ending product).

FIG. 97 is a photograph of an embodiment of a foam product of thepresent disclosure suitable for protection against UV.

FIG. 98 is a photograph of an embodiment of a viscous liquid of thepresent disclosure suitable for protection against UV.

FIG. 99 is a photograph of an embodiment of a viscous liquid of thepresent disclosure suitable for protection against U.

FIG. 100 is a photograph an embodiment of a foam product of the presentdisclosure suitable for protection against UV.

While the above-identified drawings set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION

Provided herein are methods for producing pure and highly scalable silkprotein fragment (SPF) mixture solutions that may be used acrossmultiple industries for a variety of applications. The solutions aregenerated from raw pure intact silk protein material and processed inorder to remove any sericin and achieve the desired weight averagemolecular weight (MW) and polydispersity of the fragment mixture. Selectmethod parameters may be altered to achieve distinct final silk proteinfragment characteristics depending upon the intended use. The resultingfinal fragment solution is pure silk protein fragments and water withPPM to non-detectable levels of process contaminants, levels acceptablein the pharmaceutical, medical and consumer cosmetic markets. Theconcentration, size and polydispersity of silk protein fragments in thesolution may further be altered depending upon the desired use andperformance requirements. In an embodiment, the pure silk fibroin-basedprotein fragments in the solution are substantially devoid of sericin,have an average weight average molecular weight ranging from about 6 kDato about 16 kDa, and have a polydispersity ranging from about 1.5 andabout 3.0. In an embodiment, the pure silk fibroin-based proteinfragments in the solution are substantially devoid of sericin, have anaverage weight average molecular weight ranging from about 17 kDa toabout 38 kDa, and have a polydispersity ranging from about 1.5 and about3.0. In an embodiment, the pure silk fibroin-based protein fragments inthe solution are substantially devoid of sericin, have an average weightaverage molecular weight ranging from about 39 kDa to about 80 kDa, andhave a polydispersity ranging from about 1.5 and about 3.0.

In an embodiment, the silk solutions of the present disclosure may beused to generate articles, such as silk films of various shapes andsizes by varying water content/concentration, or sold as a rawingredient into the medical, consumer, or electronics markets. In anembodiment, the solutions may be used to generate articles, such as silkgels of varying gel and liquid consistencies by varying watercontent/concentration, or sold as a raw ingredient into thepharmaceutical, medical, consumer, or electronics markets. Depending onthe silk solution utilized and the methods for casting the films orgels, various properties are achieved. The articles may be loaded withat least one therapeutic agent and/or at least one molecule.

As used herein, the terms “substantially sericin free” or “substantiallydevoid of sericin” refer to silk fibers in which a majority of thesericin protein has been removed. In an embodiment, silk fibroin that issubstantially devoid of sericin refers to silk fibroin having betweenabout 0.01% (w/w) and about 10.0% (w/w) sericin. In an embodiment, silkfibroin that is substantially devoid of sericin refers to silk fibroinhaving between about 0.01% (w/w) and about 9.0% (w/w) sericin. In anembodiment, silk fibroin that is substantially devoid of sericin refersto silk fibroin having between about 0.01% (w/w) and about 8.0% (w/w)sericin. In an embodiment, silk fibroin that is substantially devoid ofsericin refers to silk fibroin having between about 0.01% (w/w) andabout 7.0% (w/w) sericin. In an embodiment, silk fibroin that issubstantially devoid of sericin refers to silk fibroin having betweenabout 0.01% (w/w) and about 6.0% (w/w) sericin. In an embodiment, silkfibroin that is substantially devoid of sericin refers to silk fibroinhaving between about 0.01% (w/w) and about 5.0% (w/w) sericin. In anembodiment, silk fibroin that is substantially devoid of sericin refersto silk fibroin having between about 0% (w/w) and about 4.0% (w/w)sericin. In an embodiment, silk fibroin that is substantially devoid ofsericin refers to silk fibroin having between about 0.05% (w/w) andabout 4.0% (w/w) sericin. In an embodiment, silk fibroin that issubstantially devoid of sericin refers to silk fibroin having betweenabout 0.1% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silkfibroin that is substantially devoid of sericin refers to silk fibroinhaving between about 0.5% (w/w) and about 4.0% (w/w) sericin. In anembodiment, silk fibroin that is substantially devoid of sericin refersto silk fibroin having between about 1.0% (w/w) and about 4.0% (w/w)sericin. In an embodiment, silk fibroin that is substantially devoid ofsericin refers to silk fibroin having between about 1.5% (w/w) and about4.0% (w/w) sericin. In an embodiment, silk fibroin that is substantiallydevoid of sericin refers to silk fibroin having between about 2.0% (w/w)and about 4.0% (w/w) sericin. In an embodiment, silk fibroin that issubstantially devoid of sericin refers to silk fibroin having betweenabout 2.5% (w/w) and about 4.0% (w/w) sericin. In an embodiment, silkfibroin that is substantially devoid of sericin refers to silk fibroinhaving a sericin content between about 0.01% (w/w) and about 0.1% (w/w).In an embodiment, silk fibroin that is substantially devoid of sericinrefers to silk fibroin having a sericin content below about 0.1% (w/w).In an embodiment, silk fibroin that is substantially devoid of sericinrefers to silk fibroin having a sericin content below about 0.05% (w/w).In an embodiment, when a silk source is added to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes, a degumming loss of about 26 wt. %to about 31 wt. % is obtained.

As used herein, the term “substantially homogeneous” may refer to puresilk fibroin-based protein fragments that are distributed in a normaldistribution about an identified molecular weight. As used herein, theterm “substantially homogeneous” may refer to an even distribution ofadditive, for example vitamin C, throughout a composition of the presentdisclosure.

As used herein, the term “substantially free of inorganic residuals”means that the composition exhibits residuals of 0.1% (w/w) or less. Inan embodiment, substantially free of inorganic residuals refers to acomposition that exhibits residuals of 0.05% (w/w) or less. In anembodiment, substantially free of inorganic residuals refers to acomposition that exhibits residuals of 0.01% (w/w) or less. In anembodiment, the amount of inorganic residuals is between 0 ppm(“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount ofinorganic residuals is ND to about 500 ppm. In an embodiment, the amountof inorganic residuals is ND to about 400 ppm. In an embodiment, theamount of inorganic residuals is ND to about 300 ppm. In an embodiment,the amount of inorganic residuals is ND to about 200 ppm. In anembodiment, the amount of inorganic residuals is ND to about 100 ppm. Inan embodiment, the amount of inorganic residuals is between 10 ppm and1000 ppm.

As used herein, the term “substantially free of organic residuals” meansthat the composition exhibits residuals of 0.1% (w/w) or less. In anembodiment, substantially free of organic residuals refers to acomposition that exhibits residuals of 0.05% (w/w) or less. In anembodiment, substantially free of organic residuals refers to acomposition that exhibits residuals of 0.01% (w/w) or less. In anembodiment, the amount of organic residuals is between 0 ppm(“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount oforganic residuals is ND to about 500 ppm. In an embodiment, the amountof organic residuals is ND to about 400 ppm. In an embodiment, theamount of organic residuals is ND to about 300 ppm. In an embodiment,the amount of organic residuals is ND to about 200 ppm. In anembodiment, the amount of organic residuals is ND to about 100 ppm. Inan embodiment, the amount of organic residuals is between 10 ppm and1000 ppm.

Compositions of the present disclosure exhibit “biocompatibility”meaning that the compositions are compatible with living tissue or aliving system by not being toxic, injurious, or physiologically reactiveand not causing immunological rejection. Such biocompatibility can beevidenced by participants topically applying compositions of the presentdisclosure on their skin for an extended period of time. In anembodiment, the extended period of time is about 3 days. In anembodiment, the extended period of time is about 7 days. In anembodiment, the extended period of time is about 14 days. In anembodiment, the extended period of time is about 21 days. In anembodiment, the extended period of time is about 30 days. In anembodiment, the extended period of time is selected from the groupconsisting of about 1 month, about 2 months, about 3 months, about 4months, about 5 months, about 6 months, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, andindefinitely.

Compositions of the present disclosure are “hypoallergenic” meaning thatthey are relatively unlikely to cause an allergic reaction. Suchhypoallergenicity can be evidenced by participants topically applyingcompositions of the present disclosure on their skin for an extendedperiod of time. In an embodiment, the extended period of time is about 3days. In an embodiment, the extended period of time is about 7 days. Inan embodiment, the extended period of time is about 14 days. In anembodiment, the extended period of time is about 21 days. In anembodiment, the extended period of time is about 30 days. In anembodiment, the extended period of time is selected from the groupconsisting of about 1 month, about 2 months, about 3 months, about 4months, about 5 months, about 6 months, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, andindefinitely.

In an embodiment, a solution of the present disclosure is contacted witha therapeutic agent and/or a molecule prior to forming the article. Inan embodiment, molecules include, but are not limited to, antioxidantsand enzymes. In an embodiment, molecules include, but are not limitedto, Selenium, Ubiquinone derivatives, Thiol-based antioxidants,Saccharide-containing antioxidants, Polyphenols, Botanical extracts,Caffeic acid, Apigenin, Pycnogenol, Resveratrol, Folic acid, Vitaminb12, Vitamin b6, Vitamin b3, Vitamin E, Vitamin C and derivativesthereof, Vitamin D, Vitamin A, Astaxathin, Lutein, Lycopene, Essentialfatty acids (omegas 3 and 6), Iron, Zinc, magnesium, Flavonoids (soy,Curcumin, Silymarin, Pycnongeol), Growth factors, aloe, hyaluronic acid,extracellular matrix proteins, cells, nucleic acids, biomarkers,biological reagents, zinc oxide, benzyol peroxide, retnoids, titanium,allergens in a known dose (for sensitization treatment), essential oilsincluding, but not limited to, lemongrass or rosemary oil, andfragrances. Therapeutic agents include, but are not limited to, smallmolecules, drugs, proteins, peptides and nucleic acids. In anembodiment, a silk film of the present disclosure includes a moleculethat is a vitamin, such as vitamin C, vitamin A and vitamin E. In anembodiment, a solution of the present disclosure is contacted with anallergen of known quantity prior to forming the article. Allergensinclude but are not limited to milk, eggs, peanuts, tree nuts, fish,shellfish, soy and wheat. Known doses of allergen loaded within a silkarticle can be released at a known rate for controlled exposure allergystudy, tests and sensitization treatment.

In an embodiment, a solution of the present disclosure is used to createan article with microneedles by standard methods known to one in the artfor controlled delivery of molecules or therapeutic agents to or throughthe skin.

As used herein, the term “fibroin” includes silkworm fibroin and insector spider silk protein. In an embodiment, fibroin is obtained fromBombyx mori.

FIG. 1 is a flow chart showing various embodiments for producing puresilk fibroin-based protein fragments (SPFs) of the present disclosure.It should be understood that not all of the steps illustrated arenecessarily required to fabricate all silk solutions of the presentdisclosure. As illustrated in FIG. 1, step A, cocoons (heat-treated ornon-heat-treated), silk fibers, silk powder or spider silk can be usedas the silk source. If starting from raw silk cocoons from Bombyx mori,the cocoons can be cut into small pieces, for example pieces ofapproximately equal size, step B1. The raw silk is then extracted andrinsed to remove any sericin, step C1a. This results in substantiallysericin free raw silk. In an embodiment, water is heated to atemperature between 84° C. and 100° C. (ideally boiling) and then Na₂CO₃(sodium carbonate) is added to the boiling water until the Na₂CO₃ iscompletely dissolved. The raw silk is added to the boiling water/Na₂CO₃(100° C.) and submerged for approximately 15-90 minutes, where boilingfor a longer time results in smaller silk protein fragments. In anembodiment, the water volume equals about 0.4× raw silk weight and theNa₂CO₃ volume equals about 0.848× raw silk weight. In an embodiment, thewater volume equals 0.1× raw silk weight and the Na₂CO₃ volume ismaintained at 2.12 g/L. This is demonstrated in FIG. 62A and FIG. 62B:silk mass (x-axis) was varied in the same volume of extraction solution(i.e., the same volume of water and concentration of Na₂CO₃) achievingsericin removal (substantially sericin free) as demonstrated by anoverall silk mass loss of 26 to 31 percent (y-axis). Subsequently, thewater dissolved Na₂CO₃ solution is drained and excess water/Na₂CO₃ isremoved from the silk fibroin fibers (e.g., ring out the fibroin extractby hand, spin cycle using a machine, etc.). The resulting silk fibroinextract is rinsed with warm to hot water to remove any remainingadsorbed sericin or contaminate, typically at a temperature range ofabout 40° C. to about 80° C., changing the volume of water at least once(repeated for as many times as required). The resulting silk fibroinextract is a substantially sericin-depleted silk fibroin. In anembodiment, the resulting silk fibroin extract is rinsed with water at atemperature of about 60° C. In an embodiment, the volume of rinse waterfor each cycle equals 0.1 L to 0.2 L× raw silk weight. It may beadvantageous to agitate, turn or circulate the rinse water to maximizethe rinse effect. After rinsing, excess water is removed from theextracted silk fibroin fibers (e.g., ring out fibroin extract by hand orusing a machine). Alternatively, methods known to one skilled in the artsuch as pressure, temperature, or other reagents or combinations thereofmay be used for the purpose of sericin extraction. Alternatively, thesilk gland (100% sericin free silk protein) can be removed directly froma worm. This would result in liquid silk protein, without any alterationof the protein structure, free of sericin.

The extracted fibroin fibers are then allowed to dry completely. FIG. 3is a photograph showing dry extracted silk fibroin. Once dry, theextracted silk fibroin is dissolved using a solvent added to the silkfibroin at a temperature between ambient and boiling, step C1b. In anembodiment, the solvent is a solution of Lithium bromide (LiBr) (boilingfor LiBr is 140° C.). Alternatively, the extracted fibroin fibers arenot dried but wet and placed in the solvent; solvent concentration canthen be varied to achieve similar concentrations as to when adding driedsilk to the solvent. The final concentration of LiBr solvent can rangefrom 0.1M to 9.3M. FIG. 63 is a table summarizing the Molecular Weightsof silk dissolved from different concentrations of Lithium Bromide(LiBr) and from different extraction and dissolution sizes. Completedissolution of the extracted fibroin fibers can be achieved by varyingthe treatment time and temperature along with the concentration ofdissolving solvent. Other solvents may be used including, but notlimited to, phosphate phosphoric acid, calcium nitrate, calcium chloridesolution or other concentrated aqueous solutions of inorganic salts. Toensure complete dissolution, the silk fibers should be fully immersedwithin the already heated solvent solution and then maintained at atemperature ranging from about 60° C. to about 140° C. for 1-168 hrs. Inan embodiment, the silk fibers should be fully immersed within thesolvent solution and then placed into a dry oven at a temperature ofabout 100° C. for about 1 hour.

The temperature at which the silk fibroin extract is added to the LiBrsolution (or vice versa) has an effect on the time required tocompletely dissolve the fibroin and on the resulting molecular weightand polydispersity of the final SPF mixture solution. In an embodiment,silk solvent solution concentration is less than or equal to 20% w/v. Inaddition, agitation during introduction or dissolution may be used tofacilitate dissolution at varying temperatures and concentrations. Thetemperature of the LiBr solution will provide control over the silkprotein fragment mixture molecular weight and polydispersity created. Inan embodiment, a higher temperature will more quickly dissolve the silkoffering enhanced process scalability and mass production of silksolution. In an embodiment, using a LiBr solution heated to atemperature between 80° C.-140° C. reduces the time required in an ovenin order to achieve full dissolution. Varying time and temperature at orabove 60° C. of the dissolution solvent will alter and control the MWand polydispersity of the SPF mixture solutions formed from the originalmolecular weight of the native silk fibroin protein.

Alternatively, whole cocoons may be placed directly into a solvent, suchas LiBr, bypassing extraction, step B2. This requires subsequentfiltration of silk worm particles from the silk and solvent solution andsericin removal using methods know in the art for separating hydrophobicand hydrophilic proteins such as a column separation and/orchromatography, ion exchange, chemical precipitation with salt and/orpH, and or enzymatic digestion and filtration or extraction, all methodsare common examples and without limitation for standard proteinseparation methods, step C2. Non-heat treated cocoons with the silkwormremoved, may alternatively be placed into a solvent such as LiBr,bypassing extraction. The methods described above may be used forsericin separation, with the advantage that non-heat treated cocoonswill contain significantly less worm debris.

Dialysis may be used to remove the dissolution solvent from theresulting dissolved fibroin protein fragment solution by dialyzing thesolution against a volume of water, step E1. Pre-filtration prior todialysis is helpful to remove any debris (i.e., silk worm remnants) fromthe silk and LiBr solution, step D. In one example, a 3 μm or 5 μmfilter is used with a flow-rate of 200-300 mL/min to filter a 0.1% to1.0% silk-LiBr solution prior to dialysis and potential concentration ifdesired. A method disclosed herein, as described above, is to use timeand/or temperature to decrease the concentration from 9.3M LiBr to arange from 0.1M to 9.3M to facilitate filtration and downstreamdialysis, particularly when considering creating a scalable processmethod. Alternatively, without the use of additional time or temperate,a 9.3M LiBr-silk protein fragment solution may be diluted with water tofacilitate debris filtration and dialysis. The result of dissolution atthe desired time and temperate filtration is a translucent particle-freeroom temperature shelf-stable silk protein fragment-LiBr solution of aknown MW and polydispersity. It is advantageous to change the dialysiswater regularly until the solvent has been removed (e.g., change waterafter 1 hour, 4 hours, and then every 12 hours for a total of 6 waterchanges). The total number of water volume changes may be varied basedon the resulting concentration of solvent used for silk proteindissolution and fragmentation. After dialysis, the final silk solutionmaybe further filtered to remove any remaining debris (i.e., silk wormremnants).

Alternatively, Tangential Flow Filtration (TFF), which is a rapid andefficient method for the separation and purification of biomolecules,may be used to remove the solvent from the resulting dissolved fibroinsolution, step E2. TFF offers a highly pure aqueous silk proteinfragment solution and enables scalability of the process in order toproduce large volumes of the solution in a controlled and repeatablemanner. The silk and LiBr solution may be diluted prior to TFF (20% downto 0.1% silk in either water or LiBr). Pre-filtration as described aboveprior to TFF processing may maintain filter efficiency and potentiallyavoids the creation of silk gel boundary layers on the filter's surfaceas the result of the presence of debris particles. Pre-filtration priorto TFF is also helpful to remove any remaining debris (i.e., silk wormremnants) from the silk and LiBr solution that may cause spontaneous orlong-term gelation of the resulting water only solution, step D. TFF,recirculating or single pass, may be used for the creation of water-silkprotein fragment solutions ranging from 0.1% silk to 30.0% silk (morepreferably, 0.1%-6.0% silk). Different cutoff size TFF membranes may berequired based upon the desired concentration, molecular weight andpolydispersity of the silk protein fragment mixture in solution.Membranes ranging from 1-100 kDa may be necessary for varying molecularweight silk solutions created for example by varying the length ofextraction boil time or the time and temperate in dissolution solvent(e.g., LiBr). In an embodiment, a TFF 5 or 10 kDa membrane is used topurify the silk protein fragment mixture solution and to create thefinal desired silk-to-water ratio. As well, TFF single pass, TFF, andother methods known in the art, such as a falling film evaporator, maybe used to concentrate the solution following removal of the dissolutionsolvent (e.g., LiBr) (with resulting desired concentration ranging from0.1% to 30% silk). This can be used as an alternative to standard HFIPconcentration methods known in the art to create a water-based solution.A larger pore membrane could also be utilized to filter out small silkprotein fragments and to create a solution of higher molecular weightsilk with and/or without tighter polydispersity values. FIG. 61 is atable summarizing Molecular Weights for some embodiments of silk proteinsolutions of the present disclosure. Silk protein solution processingconditions were as follows: 100° C. extraction for 20 min, roomtemperature rinse, LiBr in 60° C. oven for 4-6 hours. TFF processingconditions for water-soluble films were as follows: 100° C. extractionfor 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min. FIGS.67-78 further demonstrate manipulation of extraction time, LiBrdissolution conditions, and TFF processing and resultant examplemolecular weights and polydispersities. These examples are not intendedto be limiting, but rather to demonstrate the potential of specifyingparameters for specific molecular weight silk fragment solutions.

An assay for LiBr and Na₂CO₃ detection was performed using an HPLCsystem equipped with evaporative light scattering detector (ELSD). Thecalculation was performed by linear regression of the resulting peakareas for the analyte plotted against concentration. More than onesample of a number of formulations of the present disclosure was usedfor sample preparation and analysis. Generally, four samples ofdifferent formulations were weighed directly in a 10 mL volumetricflask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH3.0) and kept at 2-8° C. for 2 hours with occasional shaking to extractanalytes from the film. After 2 hours the solution was diluted with 20mM ammonium formate (pH 3.0). The sample solution from the volumetricflask was transferred into HPLC vials and injected into the HPLC-ELSDsystem for the estimation of sodium carbonate and lithium bromide.

The analytical method developed for the quantitation of Na₂CO₃ and LiBrin silk protein formulations was found to be linear in the range 10-165μg/mL, with RSD for injection precision as 2% and 1% for area and 0.38%and 0.19% for retention time for sodium carbonate and lithium bromiderespectively. The analytical method can be applied for the quantitativedetermination of sodium carbonate and lithium bromide in silk proteinformulations.

The final silk protein fragment solution, as shown in FIG. 4, is puresilk protein fragments and water with PPM to undetectable levels ofparticulate debris and/or process contaminants, including LiBr andNa₂CO₃. FIG. 55 and FIG. 58 are tables summarizing LiBr and Na₂CO₃concentrations in solutions of the present disclosure. In FIG. 55, theprocessing conditions included 100° C. extraction for 60 min, 60° C.rinse, 100° C. LiBr in 100° C. oven for 60 min. TFF conditions includingpressure differential and number of dia-filtration volumes were varied.In FIG. 58, the processing conditions included 100° C. boil for 60 min,60° C. rinse, LiBr in 60° C. oven for 4-6 hours. In an embodiment, a SPFcomposition of the present disclosure is not soluble in an aqueoussolution due to the crystallinity of the protein. In an embodiment, aSPF composition of the present disclosure is soluble in an aqueoussolution. In an embodiment, the SPFs of a composition of the presentdisclosure include a crystalline portion of about two-thirds and anamorphous region of about one-third. In an embodiment, the SPFs of acomposition of the present disclosure include a crystalline portion ofabout one-half and an amorphous region of about one-half. In anembodiment, the SPFs of a composition of the present disclosure includea 99% crystalline portion and a 1% amorphous region. In an embodiment,the SPFs of a composition of the present disclosure include a 95%crystalline portion and a 5% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 90%crystalline portion and a 10% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 85%crystalline portion and a 15% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 80%crystalline portion and a 20% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 75%crystalline portion and a 25% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 70%crystalline portion and a 30% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 65%crystalline portion and a 35% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 60%crystalline portion and a 40% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 50%crystalline portion and a 50% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 40%crystalline portion and a 60% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 35%crystalline portion and a 65% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 30%crystalline portion and a 70% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 25%crystalline portion and a 75% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 20%crystalline portion and a 80% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 15%crystalline portion and a 85% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 10%crystalline portion and a 90% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 5% crystallineportion and a 90% amorphous region. In an embodiment, the SPFs of acomposition of the present disclosure include a 1% crystalline portionand a 99% amorphous region.

A unique feature of the SPF compositions of the present disclosure areshelf stability (they will not slowly or spontaneously gel when storedin an aqueous solution and there is no aggregation of fragments andtherefore no increase in molecular weight over time), from 10 days to 3years depending on storage conditions, percent silk, and number ofshipments and shipment conditions. Additionally pH may be altered toextend shelf-life and/or support shipping conditions by preventingpremature folding and aggregation of the silk. In an embodiment, a SPFsolution composition of the present disclosure has a shelf stability forup to 2 weeks at room temperature (RT). In an embodiment, a SPF solutioncomposition of the present disclosure has a shelf stability for up to 4weeks at RT. In an embodiment, a SPF solution composition of the presentdisclosure has a shelf stability for up to 6 weeks at RT. In anembodiment, a SPF solution composition of the present disclosure has ashelf stability for up to 8 weeks at RT. In an embodiment, a SPFsolution composition of the present disclosure has a shelf stability forup to 10 weeks at RT. In an embodiment, a SPF solution composition ofthe present disclosure has a shelf stability for up to 12 weeks at RT.In an embodiment, a SPF solution composition of the present disclosurehas a shelf stability ranging from about 4 weeks to about 52 weeks atRT. Table 1 below shows shelf stability test results for embodiments ofSPF compositions of the present disclosure.

TABLE 1 Shelf Stability of SPF Compositions of the Present Disclosure %Silk Temperature Time to Gelation 2 RT  4 weeks 2 4 C. >9 weeks 4 RT  4weeks 4 4 C. >9 weeks 6 RT  2 weeks 6 4 C. >9 weeks

A known additive such as a vitamin (e.g., vitamin C) can be added to aSPF composition of the present disclosure to create a gel that is stablefrom 10 days to 3 years at room temperature (RT). Both examples, a SPFcomposition and the same with an additive, can be lyophilized forenhanced storage control ranging from 10 days to 10 years depending onstorage and shipment conditions. The lyophilized silk powder can also beused as a raw ingredient in the medical, consumer, and electronicmarkets. Additionally, lyophilized silk powder can be resuspended inwater, HFIP, or organic solution following storage to create silksolutions of varying concentrations, including higher concentrationsolutions than those produced initially. In another embodiment, the silkfibroin-based protein fragments are dried using a rototherm evaporatoror other methods known in the art for creating a dry protein formcontaining less than 10% water by mass.

Either the silk fragment-water solutions or the lyophilized silk proteinfragment mixture can be sterilized following standard methods in the artnot limited to filtration, heat, radiation or e-beam. It is anticipatedthat the silk protein fragment mixture, because of its shorter proteinpolymer length, will withstand sterilization better than intact silkprotein solutions described in the art. Additionally, silk articlescreated from the SPF mixtures described herein may be sterilized asappropriate to application. For example, a silk film loaded with amolecule to be used in medical applications with an open wound/incision,may be sterilized standard methods such as by radiation or e-beam.

FIG. 2 is a flow chart showing various parameters that can be modifiedduring the process of producing a silk protein fragment solution of thepresent disclosure during the extraction and the dissolution steps.Select method parameters may be altered to achieve distinct finalsolution characteristics depending upon the intended use, e.g.,molecular weight and polydispersity. It should be understood that notall of the steps illustrated are necessarily required to fabricate allsilk solutions of the present disclosure.

In an embodiment, a process for producing a silk protein fragmentsolution of the present disclosure includes forming pieces of silkcocoons from the Bombyx mori silk worm; extracting the pieces at about100° C. in a solution of water and Na₂CO₃ for about 60 minutes, whereina volume of the water equals about 0.4× raw silk weight and the amountof Na₂CO₃ is about 0.848× the weight of the pieces to form a silkfibroin extract; triple rinsing the silk fibroin extract at about 60° C.for about 20 minutes per rinse in a volume of rinse water, wherein therinse water for each cycle equals about 0.2 L× the weight of the pieces;removing excess water from the silk fibroin extract; drying the silkfibroin extract; dissolving the dry silk fibroin extract in a LiBrsolution, wherein the LiBr solution is first heated to about 100° C. tocreate a silk and LiBr solution and maintained; placing the silk andLiBr solution in a dry oven at about 100° C. for about 60 minutes toachieve complete dissolution and further fragmentation of the nativesilk protein structure into mixture with desired molecular weight andpolydispersity; filtering the solution to remove any remaining debrisfrom the silkworm; diluting the solution with water to result in a 1%silk solution; and removing solvent from the solution using TangentialFlow Filtration (TFF). In an embodiment, a 10 kDa membrane is utilizedto purify the silk solution and create the final desired silk-to-waterratio. TFF can then be used to further concentrate the pure silksolution to a concentration of 2% silk to water.

Each process step from raw cocoons to dialysis is scalable to increaseefficiency in manufacturing. Whole cocoons are currently purchased asthe raw material, but pre-cleaned cocoons or non-heat treated cocoons,where worm removal leaves minimal debris, have also been used. Cuttingand cleaning the cocoons is a manual process, however for scalabilitythis process could be made less labor intensive by, for example, usingan automated machine in combination with compressed air to remove theworm and any particulates, or using a cutting mill to cut the cocoonsinto smaller pieces. The extraction step, currently performed in smallbatches, could be completed in a larger vessel, for example anindustrial washing machine where temperatures at or in between 60° C. to100° C. can be maintained. The rinsing step could also be completed inthe industrial washing machine, eliminating the manual rinse cycles.Dissolution of the silk in LiBr solution could occur in a vessel otherthan a convection oven, for example a stirred tank reactor. Dialyzingthe silk through a series of water changes is a manual and timeintensive process, which could be accelerated by changing certainparameters, for example diluting the silk solution prior to dialysis.The dialysis process could be scaled for manufacturing by usingsemi-automated equipment, for example a tangential flow filtrationsystem.

Varying extraction (i.e., time and temperature), LiBr (i.e., temperatureof LiBr solution when added to silk fibroin extract or vice versa) anddissolution (i.e., time and temperature) parameters results in solventand silk solutions with different viscosities, homogeneities, and colors(see FIGS. 5-32). Increasing the temperature for extraction, lengtheningthe extraction time, using a higher temperature LiBr solution atemersion and over time when dissolving the silk and increasing the timeat temperature (e.g., in an oven as shown here, or an alternative heatsource) all resulted in less viscous and more homogeneous solvent andsilk solutions. While almost all parameters resulted in a viable silksolution, methods that allow complete dissolution to be achieved infewer than 4 to 6 hours are preferred for process scalability.

FIGS. 5-10 show photographs of four different silk extractioncombinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and100° C. 60 min. Briefly, 9.3 M LiBr was prepared and allowed to sit atroom temperature for at least 30 minutes. 5 mL of LiBr solution wasadded to 1.25 g of silk and placed in the 60° C. oven. Samples from eachset were removed at 4, 6, 8, 12, 24, 168 and 192 hours. The remainingsample was photographed.

FIGS. 11-23 show photographs of four different silk extractioncombinations tested: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and100° C. 60 min. Briefly, 9.3 M LiBr solution was heated to one of fourtemperatures: 60° C., 80° C., 100° C. or boiling. 5 mL of hot LiBrsolution was added to 1.25 g of silk and placed in the 60° C. oven.Samples from each set were removed at 1, 4 and 6 hours. The remainingsample was photographed.

FIGS. 24-32 show photographs of four different silk extractioncombinations tested: Four different silk extraction combinations wereused: 90° C. 30 min, 90° C. 60 min, 100° C. 30 min, and 100° C. 60 min.Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60°C., 80° C., 100° C. or boiling. 5 mL of hot LiBr solution was added to1.25 g of silk and placed in the oven at the same temperature of theLiBr. Samples from each set were removed at 1, 4 and 6 hours. 1 mL ofeach sample was added to 7.5 mL of 9.3 M LiBr and refrigerated forviscosity testing. The remaining sample was photographed.

Molecular weight of the silk protein fragments may be controlled basedupon the specific parameters utilized during the extraction step,including extraction time and temperature; specific parameters utilizedduring the dissolution step, including the LiBr temperature at the timeof submersion of the silk in to the lithium bromide and time that thesolution is maintained at specific temperatures; and specific parametersutilized during the filtration step. By controlling process parametersusing the disclosed methods, it is possible to create SPF mixturesolutions with polydispersity equal to or lower than 2.5 at a variety ofdifferent molecular weight ranging from 5 kDa to 200 kDa, morepreferably between 10 kDa and 80 kDA. By altering process parameters toachieve silk solutions with different molecular weights, a range offragment mixture end products, with desired polydispersity of equal toor less than 2.5 may be targeted based upon the desired performancerequirements. For example, a lower molecular weight silk film containinga drug may have a faster release rate compared to a higher molecularweight film making it more ideal for a daily delivery vehicle inconsumer cosmetics. Additionally, SPF mixture solutions with apolydispersity of greater than 2.5 can be achieved. Further, twosolutions with different average molecular weights and polydispersitiescan be mixed to create combination solutions. Alternatively, a liquidsilk gland (100% sericin free silk protein) that has been removeddirectly from a worm could be used in combination with any of the SPFmixture solutions of the present disclosure. Molecular weight of thepure silk fibroin-based protein fragment composition was determinedusing High Pressure Liquid Chromatography (HPLC) with a Refractive IndexDetector (RID). Polydispersity was calculated using Cirrus GPC OnlineGPC/SEC Software Version 3.3 (Agilent).

Parameters were varied during the processing of raw silk cocoons intosilk solution. Varying these parameters affected the MW of the resultingsilk solution. Parameters manipulated included (i) time and temperatureof extraction, (ii) temperature of LiBr, (iii) temperature ofdissolution oven, and (iv) dissolution time. Molecular weight wasdetermined with mass spec as shown in FIGS. 64-80.

Experiments were carried out to determine the effect of varying theextraction time. FIGS. 64-70 are graphs showing these results, andTables 2-8 summarize the results. Below is a summary:

-   -   A sericin extraction time of 30 minutes resulted in larger MW        than a sericin extraction time of 60 minutes    -   MW decreases with time in the oven    -   140° C. LiBr and oven resulted in the low end of the confidence        interval to be below a MW of 9500 Da    -   30 min extraction at the 1 hour and 4 hour time points have        undigested silk    -   30 min extraction at the 1 hour time point resulted in a        significantly high molecular weight with the low end of the        confidence interval being 35,000 Da    -   The range of MW reached for the high end of the confidence        interval was 18000 to 216000 Da (important for offering        solutions with specified upper limit)

TABLE 2 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 100° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average Std ConfidenceTime Time Mw dev Interval PD 30 1 57247 12780 35093 93387 1.63 60 131520 1387 11633 85407 2.71 30 4 40973 2632 14268 117658 2.87 60 4 250821248 10520 59803 2.38 30 6 25604 1405 10252 63943 2.50 60 6 20980 126210073 43695 2.08

TABLE 3 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, boiling Lithium Bromide (LiBr) and 60° C. Oven Dissolutionfor 4 hr. Boil Average Std Confidence Sample Time Mw dev Interval PD 30min, 4 hr 30 49656 4580 17306 142478 2.87 60 min, 4 hr 60 30042 153611183 80705 2.69

TABLE 4 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 60° C. Lithium Bromide (LiBr) and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average Std ConfidenceSample Time Time Mw dev Interval PD 30 min, 1 hr 30 1 58436 22201 1538092.63 60 min, 1 hr 60 1 31700 11931 84224 2.66 30 min, 4 hr 30 4 61956.513337 21463 178847 2.89 60 min, 4 hr 60 4 25578.5 2446 9979 65564 2.56

TABLE 5 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 80° C. Lithium Bromide (LiBr) and 80° C. Oven Dissolutionfor 6 hr. Average Std Confidence Sample Boil Time Mw dev Interval PD 30min, 6 hr 30 63510 18693 215775 3.40 60 min, 6 hr 60 25164 238 963765706 2.61

TABLE 6 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 80° C. Lithium Bromide (LiBr) and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average Confidence SampleTime Time Mw Std dev Interval PD 30 min, 4 hr 30 4 59202 14028 19073183760 3.10 60 min, 4 hr 60 4 26312.5 637 10266 67442 2.56 30 min, 6 hr30 6 46824 18076 121293 2.59 60 min, 6 hr 60 6 26353 10168 68302 2.59

TABLE 7 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average Confidence SampleTime Time Mw Std dev Interval PD 30 min, 4 hr 30 4 47853 19758 1159002.42 60 min, 4 hr 60 4 25082 1248 10520 59804 2.38 30 min, 6 hr 30 655421 8992 19153 160366 2.89 60 min, 6 hr 60 6 20980 1262 10073 436942.08

TABLE 8 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 140° C. Lithium Bromide (LiBr) and 140° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average Confidence SampleTime Time Mw Std dev Interval PD 30 min, 4 hr 30 4 9024.5 1102 449318127 2.00865 60 min, 4 hr 60 4 15548 6954 34762 2.2358 30 min, 6 hr 306 13021 5987 28319 2.1749 60 min, 6 hr 60 6 10888 5364 22100 2.0298

Experiments were carried out to determine the effect of varying theextraction temperature. FIG. 71 is a graph showing these results, andTable 9 summarizes the results. Below is a summary:

-   -   Sericin extraction at 90° C. resulted in higher MW than sericin        extraction at 100° C. extraction    -   Both 90° C. and 100° C. show decreasing MW over time in the oven

TABLE 9 The effect of extraction temperature (90° C. vs. 100° C.) onmolecular weight of silk processed under the conditions of 60 min.Extraction Temperature, 100° C. Lithium Bromide (LiBr) and 100° C. OvenDissolution (Oven/Dissolution Time was varied). Boil Oven Average StdConfidence Sample Time Time Mw dev Interval PD  90 C., 4 hr 60 4 373084204 13368 104119 2.79 100 C., 4 hr 60 4 25082 1248 10520 59804 2.38  90C., 6 hr 60 6 34224 1135 12717 92100 2.69 100 C., 6 hr 60 6 20980 126210073 43694 2.08

Experiments were carried out to determine the effect of varying theLithium Bromide (LiBr) temperature when added to silk. FIGS. 72-73 aregraphs showing these results, and Tables 10-11 summarize the results.Below is a summary:

-   -   No impact on MW or confidence interval (all CI˜10500-6500 Da)    -   Studies illustrated that the temperature of LiBr-silk        dissolution, as LiBr is added and begins dissolving, rapidly        drops below the original LiBr temperature due to the majority of        the mass being silk at room temp

TABLE 10 The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 60 min. ExtractionTime., 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). LiBr Temp Oven Average StdConfidence Sample (° C.) Time Mw dev Interval PD 60 C. LiBr, 60 1 3170011931 84223 2.66 1 hr 100 C. LiBr, 100 1 27907 200 10735 72552 2.60 1 hrRT LiBr, 4 hr RT 4 29217 1082 10789 79119 2.71 60 C. LiBr, 60 4 255782445 9978 65564 2.56 4 hr 80 C. LiBr, 80 4 26312 637 10265 67441 2.56 4hr 100 C. LiBr, 100 4 27681 1729 11279 67931 2.45 4 hr Boil LiBr, 4 hrBoil 4 30042 1535 11183 80704 2.69 RT LiBr, 6 hr RT 6 26543 1893 1078365332 2.46 80 C. LiBr, 80 6 26353 10167 68301 2.59 6 hr 100 C. LiBr, 1006 27150 916 11020 66889 2.46 6 hr

TABLE 11 The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 30 min. ExtractionTime, 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). LiBr Temp Oven Average StdConfidence Sample (° C.) Time Mw dev Interval PD 60 C. LiBr, 60 4 6195613336 21463 178847 2.89 4 hr 80 C. LiBr, 80 4 59202 14027 19073 1837603.10 4 hr 100 C. LiBr, 100 4 47853 19757 115899 2.42 4 hr 80 C. LiBr, 806 46824 18075 121292 2.59 6 hr 100 C. LiBr, 100 6 55421 8991 19152160366 2.89 6 hr

Experiments were carried out to determine the effect of voven/dissolution temperature. FIGS. 74-78 are graphs showing theseresults, and Tables 12-16 summarize the results. Below is a summary:

-   -   Oven temperature has less of an effect on 60 min extracted silk        than 30 min extracted silk. Without wishing to be bound by        theory, it is believed that the min silk is less degraded during        extraction and therefore the oven temperature has more of an        effect on the larger MW, less degraded portion of the silk.    -   For 60° C. vs. 140° C. oven the 30 min extracted silk showed a        very significant effect of lower MW at higher oven temp, while        60 min extracted silk had an effect but much less    -   The 140° C. oven resulted in a low end in the confidence        interval at ˜6000 Da

TABLE 12 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 100° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven AverageConfidence Time (° C.) Time Mw Std dev Interval PD 30 60 4 47853 19758115900 2.42 30 100 4 40973 2632 14268 117658 2.87 30 60 6 55421 899219153 160366 2.89 30 100 6 25604 1405 10252 63943 2.50

TABLE 13 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 100° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven AverageConfidence Time (° C.) Time Mw Std dev Interval PD 60 60 1 27908 20010735 72552 2.60 60 100 1 31520 1387 11633 85407 2.71 60 60 4 27681 173011279 72552 2.62 60 100 4 25082 1248 10520 59803 2.38 60 60 6 27150 91611020 66889 2.46 60 100 6 20980 1262 10073 43695 2.08

TABLE 14 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 140° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Oven Average ConfidenceTime Temp (° C.) Time Mw Std dev Interval PD 60 60 4 30042 1536 1118380705 2.69 60 140 4 15548 7255 33322 2.14

TABLE 15 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 140° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Oven Temp Oven Average ConfidenceBoil Time (° C.) Time Mw Std dev Interval PD 30 60 4 49656 4580 17306142478 2.87 30 140 4 9025 1102 4493 18127 2.01 30 60 6 59383 11640 17641199889 3.37 30 140 6 13021 5987 28319 2.17

TABLE 16 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 80° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven AverageConfidence Time (° C.) Time Mw Std dev Interval PD 60 60 4 26313 63710266 67442 2.56 60 80 4 30308 4293 12279 74806 2.47 60 60 6 26353 1016868302 2.59 60 80 6 25164 238 9637 65706 2.61

In an embodiment, the methods disclosed herein result in a solution withcharacteristics that can be controlled during manufacturing, including,but not limited to: MW—may be varied by changing extraction and/ordissolution time and temp (e.g., LiBr temperature), pressure, andfiltration (e.g., size exclusion chromatography); Structure—removal orcleavage of heavy or light chain of the fibroin protein polymer;Purity—hot water rinse temperature for improved sericin removal orfilter capability for improved particulate removal that adverselyaffects shelf stability of the silk fragment protein mixture solution;Color—the color of the solution can be controlled with, for example,LiBr temp and time; Viscosity; Clarity; and Stability of solution. Theresultant pH of the solution is typically about 7 and can be alteredusing an acid or base as appropriate to storage requirements.

The above-described SPF mixture solutions may be utilized to produce apure silk protein fragment-film or pure silk protein fragment-gel fornumerous applications (e.g., delivery of a drug, vitamin, antioxidant,etc. to the skin). FIG. 33 is a flow chart showing an embodiment forproducing a silk film of the present disclosure from a silk solution ofthe present disclosure. In step A, a silk solution of the presentdisclosure is chosen, and then at least on molecule or therapeutic agentis added directly to the silk solution prior to gel or film processing,step B. When producing a silk film, the silk solution with additive(s)may be cast directly onto a shaped mold to achieve a unique film shape(e.g., silicone mold) or the silk solution may be cast as a sheet andthen subsequently cut or punched into a variety of shapes, with avariety of cutting techniques, including, but not limited to cuttingwith a rotary blade or laser cutting for example (FIGS. 83A and 83B),depending upon the desired application, step C. If cast on a mold, forexample silicone, the silicone mold may be heated on alaser-etched/patterned surface to create an impression that will betransferred to the final film. For example, the product logo could betransferred to the film, visible, but not palpable by hand, and used toshow authenticity of the product. The concentration and/or mass of thefinal silk protein fragment film can be varied to control the film'sdegree of flexibility and conformity to different anatomicaltopographies. Altering the drying method for a silk film will alsoresult in different final film characteristics. Applying airflow and/orheat impacts the properties of the film (e.g., brittleness, number ofbubbles, curling, solubility, surface appearance), step D. Additionally,the percent moisture within the film at the time of packaging willimpact stability over time with too much moisture resulting in yellowingof the films with time (FIGS. 82A-82C). In some embodiments, filmsideally may have between about 2 to about 20% water content atcompletion of drying. It was observed that greater moisture content than20% in the films will decrease shelf life. If films are not dry enough(that is they have greater than 20% water content) before packaging,they will yellow over time (2+ weeks). It is advised that films aredried in an incubator until the relative humidity in the incubator isless than the relative humidity in the surrounding area and no greaterthan 36%. Ambient humidity will have an effect on the ability to removemoisture and therefore, a tactile/audio test can be used to determinewhether films are ready for packaging. In an embodiment, the testincludes removal of a film from the drying system, slightly bending oneend of the film and releasing it. If the film feels and sounds similarto a piece of paper or thin plastic, it is considered dry. If the filmhas not completed drying, it will be pliable and will make no noise uponbending and release. In an embodiment, the film is flexible without theneed for process additives such as glycerin, such that a film that is2.5 cm wide by 10 cm long can be bent in half so that opposite ends ofthe film can touch one another without the film breaking or cracking Afilm of this same size can be bent in half along the length of the filmto create a 45-degree angle without breaking or cracking the film.

The final silk protein fragment-film is pure with undetectable levels ofparticulate debris and/or process contaminants, including LiBr andNa₂CO₃. Alternatively, the final SPF mixture solution has less than 500ppm process contaminants. FIG. 56 and FIG. 57 are tables summarizingLiBr and Na₂CO₃ concentrations in films (2% silk films air dried at RT)of the present disclosure. In FIG. 56, the processing conditionsincluded 100° C. extraction for 20 min, RT rinse, LiBr in 60° C. ovenfor 4-6 hours. In FIG. 57, the processing conditions included 100° C.extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours.

In an embodiment, when producing a silk gel, an acid is used to helpfacilitate gelation. In an embodiment, when producing a silk gel thatincludes a neutral or a basic molecule and/or therapeutic agent, an acidcan be added to facilitate gelation. In an embodiment, when producing asilk gel, increasing the pH (making the gel more basic) increases theshelf stability of the gel. In an embodiment, when producing a silk gel,increasing the pH (making the gel more basic) allows for a greaterquantity of an acidic molecule to be loaded into the gel.

In an embodiment, natural additives may be added to the silk gel tofurther stabilize additives. For example, trace elements such asselenium or magnesium or L-methoinine can be used. Further, light-blockcontainers can be added to further increase stability.

FIG. 34 summarizes an embodiment of parameters for a silk fragment-filmdrying study of the present disclosure. FIG. 35 is a graph showing silkfragment-film drying times (under various air flow and temperatureconditions) based on the silk fragment-film drying study of FIG. 34.These studies indicate that airflow is an important parameter toconsider for drying (i.e., samples in covered containers did not dry),temperature can be altered to alter drying rate (i.e., increasedtemperature results in a faster rate of water removal) and that asteady-state of moisture content within the films can be obtained with avariety of parameters (i.e., from 24 to 48 hours, mass is consistent inuncovered samples regardless of temperature). Of note, the finalproperties of the film, for example brittleness, will vary with dryingconditions. Alternatively, film drying rate may be accelerated by theuse of an additive in the SPF solution, such as a surfactant or oil.These additives may be used with or without heat to alter drying rateand final film physical properties.

In an embodiment, the drying conditions of the SFP film are 24° C. in aforced air flow incubator for 12 to 48 hours depending on the number offilms and ambient humidity. Under these drying conditions, a film thatwill not shrink more than 5 percent over time when stored in a foilpouch is created. Additionally, the film is homogeneous in compositionand physical structure, with no sided-ness and an even distribution ofadditive, for example vitamin C, throughout.

In an embodiment, the silk protein fragment-film may stabilize vitamin Cand derivatives thereof at room temperature when stored in lightretaining about 30% to about 100% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 35% to about 95% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 40% to about 90% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 45% to about 85% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 50% to about 80% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 55% to about 75% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored inlight retaining about 60% to about 70% of its activity after 30 days ofstorage. In an embodiment, the silk protein fragment-film may stabilizevitamin C and derivatives thereof at room temperature when stored in asealed airtight container or pouch that prevents light from contactingthe film retaining about 80% to about 100% of its activity after 3 to 24months of storage. In an embodiment, the silk protein fragment-film maystabilize vitamin C and derivatives thereof at room temperature whenstored in a sealed airtight container or pouch that prevents light fromcontacting the film retaining about 80% to about 100% of its activityafter about 3 to about 60 months of storage. In an embodiment, the silkprotein fragment-film may release between 50% to 90% of active vitamin Cand derivatives thereof within 20 mins when adhered to dampened skin. Inan embodiment, the silk protein fragment-film may release at least 50%active vitamin C and derivatives thereof within 20 mins when adhered todampened skin. In an embodiment, the silk protein fragment-film mayrelease at least 60% active vitamin C and derivatives thereof within 20mins when adhered to dampened skin. In an embodiment, the silk proteinfragment-film may release at least 70% active vitamin C and derivativesthereof within 20 mins when adhered to dampened skin. In an embodiment,the silk protein fragment-film may release at least 80% active vitamin Cand derivatives thereof within 20 mins when adhered to dampened skin. Inan embodiment, the silk protein fragment-film may release at least 90%active vitamin C and derivatives thereof within 20 mins when adhered todampened skin. In an embodiment, the silk protein fragment-film mayrelease between 10% to 100% of active vitamin C and derivatives thereofwithin 5 mins to 8 hours when adhered to dampened skin. In anembodiment, the silk protein fragment-film may release at least 10% ofactive vitamin C and derivatives thereof within 5 mins to 8 hours whenadhered to dampened skin. In an embodiment, the silk proteinfragment-film may release at least 20% of active vitamin C andderivatives thereof within 5 mins to 8 hours when adhered to dampenedskin. In an embodiment, the silk protein fragment-film may release atleast 30% of active vitamin C and derivatives thereof within 5 mins to 8hours when adhered to dampened skin. In an embodiment, the silk proteinfragment-film may release at least 40% of active vitamin C andderivatives thereof within 5 mins to 8 hours when adhered to dampenedskin. In an embodiment, the silk protein fragment-film may release atleast 50% of active vitamin C and derivatives thereof within 5 mins to 8hours when adhered to dampened skin. In an embodiment, the silk proteinfragment-film may release at least 60% of active vitamin C andderivatives thereof within 5 mins to 8 hours when adhered to dampenedskin. In an embodiment, the silk protein fragment-film may release atleast 70% of active vitamin C and derivatives thereof within 5 mins to 8hours when adhered to dampened skin. In an embodiment, the silk proteinfragment-film may release at least 80% of active vitamin C andderivatives thereof within 5 mins to 8 hours when adhered to dampenedskin. In an embodiment, the silk protein fragment-film may release atleast 90% of active vitamin C and derivatives thereof within 5 mins to 8hours when adhered to dampened skin. It is believed that exposure tohigher temperatures for a longer period of time may break down the silkprotein into more versatile silk protein fragment mixtures and/ordisrupt any silk protein tertiary and/or secondary silk proteinstructure that could adversely affect shelf stability and/or performanceof resulting structures (e.g., gels, films, foams, etc.) as well asreduces the number of heavy chains within the silk protein.

FIGS. 36A and 36B show two HPLC chromatograms from samples comprisingvitamin C. The chromatogram on the left shows peaks from (1) achemically stabilized sample of vitamin C at ambient conditions and (2)a sample of vitamin C taken after 1 hour at ambient conditions withoutchemical stabilization to prevent oxidation, where degradation productsare visible. The chromatogram on the right shows peaks from twodifferent embodiments of silk films of the present disclosure that wereaged for at least 30 days at room temperature. No degradation productswere visible. FIG. 59 is a table summarizing the vitamin C concentrationin silk protein fragment-films (2% silk films air dried at RT) of thepresent disclosure. In FIG. 59 processing conditions included 100° C.extraction for 20 min, RT rinse, LiBr in 60° C. oven for 4-6 hours. FIG.60 is a table summarizing the stability of vitamin C in chemicallystabilized solutions. FIGS. 89A-89B are tables summarizing vitamin Cstability in SPF gels without chemical stabilizers as compared tochemically stabilized vitamin C in competitive anti-aging skincareproducts. A gel cast at 20% total vitamin C additive concentration didnot gel. Without wishing to be bound by theory, it appears there is arelationship between vitamin C concentration, silk concentration, andgelation. An increase in vitamin C at a given concentration of silk willresult in a longer time to gelation or inhibit gelation. This may be dueto the vitamin C molecule physically blocking interaction between silkprotein fragments or cross-linking of silk protein.

In an embodiment, the molecule or molecules are stable and can bereleased over an extended time period. In an embodiment, release rate iscontrolled by the specific weight average molecular weight of the silkfibroin-based protein fragments used. In another embodiment, releaserate is controlled by creation of a multi-layer structure. For example,multiple films can be cast and dried upon each other. Additionally, eachlayer can be formed using the same or different molecular weightcompositions. In an embodiment, the degree of crystallinity of theprotein structure is altered through film drying conditions, therebycontrolling the release rate. The molecule or molecules may be releasedtopically on the skin, subcutaneously following implantation, or locallyor systemically through oral administration or implantation. In anembodiment, the molecule or molecules is released between 1 minutes and20 minutes. In an embodiment, the molecule or molecules is releasedbetween 20 minutes and 60 minutes. In an embodiment, the molecule ormolecules is released between 1 hour and 4 hours. In an embodiment, themolecule or molecules is released between 4 hours and 8 hours. In anembodiment, the molecule or molecules is released between 8 hours and 24hours. In an embodiment, the molecule or molecules is released between 1day and 7 days. In an embodiment, the molecule or molecules is releasedbetween 1 week and 4 weeks. In an embodiment, the molecule or moleculesis released between 1 month and 3 months. In an embodiment, the moleculeor molecules is released between 3 months and 6 months. In anembodiment, the molecule or molecules is released between 20 minutes and6 months. In an embodiment, the molecule or molecules are stable atextreme temperature and humidity conditions.

Films of the present disclosure comprised of about 20 kDA average weightaverage molecular weight silk fibroin-based protein fragments andcontaining about 20% vitamin C by mass, were stored individually withinfoil pouches and exposed to temperature extremes. Foil pouchescontaining films were exposed to:

-   -   Ambient conditions (time 0 films)    -   “Extreme Cold” (−29° C.±2° C. for 72 hours), followed by “Hot        Humid” (38° C.±2° C. at 85% Humidity±5% for 72 hours), and        subsequently “Extreme Heat, Moderate Humidity” (60° C.±2° C. at        30% Humidity±5% for 6 hours)

The amount of active vitamin C was measured using HPLC. All films wereobserved to support maintenance of vitamin C activity with exposure toextremes, as summarized in Table 17.

TABLE 17 Amount of active vitamin C in films under varying conditionsAverage Conc of vit C in sample N Conditions (mg/g) Std. Dev 4 Time 0,ambient conditions 184.90 15.15 16 1) −29° C. ± 2° C. for 72 hours193.97 10.25 2) 38° C. ± 2° C. at 85% Humidity ± 5% for 72 hours 3) 60°C. ± 2° C. at 30% Humidity ± 5% for 6 hours

FIGS. 37-45 are photographs showing silk protein fragment-films of thepresent disclosure dried under various temperature, time and dryingconditions.

FIGS. 46-54 are photographs showing the dissolution, in water, of theformed silk protein fragment-films of the present disclosure undervarious temperature, time and drying conditions. The water solubility offilms of the present disclosure may be varied by altering dryingconditions. For example, drying a film to 20% humidity in a forced airincubator and then increasing ambient humidity to 50% for a period ofhours and subsequently drying the film back to 20% humidity will resultin an insoluble film. Under ordinary conditions where the humidity issteadily decreased, a water-soluble silk film is created. It isanticipated that the increase in humidity allowed the protein structureto be further mobilized in the film and further crystallized, resultingin a non-soluble film. Alternative methods in the art to createnon-soluble films include the introduction of methanol. The films of thepresent disclosure are clearly differentiated from those films due totheir solubility in water. The SFP gel articles described herein rangefrom a hydrogel which can be injected or spread topically to a film-gelarticle that appears as a film and contains a minimal but controlledwater content, thereby preventing crystallinity and allowing watersolubility.

In some embodiments, a composition of the present disclosure can furtherinclude skin penetration enhancers, including, but not limited to,sulfoxides (such as dimethylsulfoxide), pyrrolidones (such as2-pyrrolidone), alcohols (such as ethanol or decanol), azones (such aslaurocapram and 1-dodecylazacycloheptan-2-one), surfactants (includingalkyl carboxylates and their corresponding acids such as oleic acid,fluoroalkylcarboxylates and their corresponding acids, alkyl sulfates,alkyl ether sulfates, docusates such as dioctyl sodium sulfosuccinate,alkyl benzene sulfonates, alkyl ether phosphates, and alkyl aryl etherphosphates), glycols (such as propylene glycol), terpenes (such aslimonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol,carveol, carvone, fenchone, and verbenone), and dimethyl isosorbide.

Following are non-limiting examples of suitable ranges for variousparameters in and for preparation of the silk solutions of the presentdisclosure. The silk solutions of the present disclosure may include oneor more, but not necessarily all, of these parameters and may beprepared using various combinations of ranges of such parameters.

In an embodiment, the percent silk in the solution is less than 30%. Inan embodiment, the percent silk in the solution is less than 25%. In anembodiment, the percent silk in the solution is less than 20%. In anembodiment, the percent silk in the solution is less than 19%. In anembodiment, the percent silk in the solution is less than 18%. In anembodiment, the percent silk in the solution is less than 17%. In anembodiment, the percent silk in the solution is less than 16%. In anembodiment, the percent silk in the solution is less than 15%. In anembodiment, the percent silk in the solution is less than 14%. In anembodiment, the percent silk in the solution is less than 13%. In anembodiment, the percent silk in the solution is less than 12%. In anembodiment, the percent silk in the solution is less than 11%. In anembodiment, the percent silk in the solution is less than 10%. In anembodiment, the percent silk in the solution is less than 9%. In anembodiment, the percent silk in the solution is less than 8%. In anembodiment, the percent silk in the solution is less than 7%. In anembodiment, the percent silk in the solution is less than 6%. In anembodiment, the percent silk in the solution is less than 5%. In anembodiment, the percent silk in the solution is less than 4%. In anembodiment, the percent silk in the solution is less than 3%. In anembodiment, the percent silk in the solution is less than 2%. In anembodiment, the percent silk in the solution is less than 1%. In anembodiment, the percent silk in the solution is less than 0.9%. In anembodiment, the percent silk in the solution is less than 0.8%. In anembodiment, the percent silk in the solution is less than 0.7%. In anembodiment, the percent silk in the solution is less than 0.6%. In anembodiment, the percent silk in the solution is less than 0.5%. In anembodiment, the percent silk in the solution is less than 0.4%. In anembodiment, the percent silk in the solution is less than 0.3%. In anembodiment, the percent silk in the solution is less than 0.2%. In anembodiment, the percent silk in the solution is less than 0.1%. In anembodiment, the percent silk in the solution is greater than 0.1%. In anembodiment, the percent silk in the solution is greater than 0.2%. In anembodiment, the percent silk in the solution is greater than 0.3%. In anembodiment, the percent silk in the solution is greater than 0.4%. In anembodiment, the percent silk in the solution is greater than 0.5%. In anembodiment, the percent silk in the solution is greater than 0.6%. In anembodiment, the percent silk in the solution is greater than 0.7%. In anembodiment, the percent silk in the solution is greater than 0.8%. In anembodiment, the percent silk in the solution is greater than 0.9%. In anembodiment, the percent silk in the solution is greater than 1%. In anembodiment, the percent silk in the solution is greater than 2%. In anembodiment, the percent silk in the solution is greater than 3%. In anembodiment, the percent silk in the solution is greater than 4%. In anembodiment, the percent silk in the solution is greater than 5%. In anembodiment, the percent silk in the solution is greater than 6%. In anembodiment, the percent silk in the solution is greater than 7%. In anembodiment, the percent silk in the solution is greater than 8%. In anembodiment, the percent silk in the solution is greater than 9%. In anembodiment, the percent silk in the solution is greater than 10%. In anembodiment, the percent silk in the solution is greater than 11%. In anembodiment, the percent silk in the solution is greater than 12%. In anembodiment, the percent silk in the solution is greater than 13%. In anembodiment, the percent silk in the solution is greater than 14%. In anembodiment, the percent silk in the solution is greater than 15%. In anembodiment, the percent silk in the solution is greater than 16%. In anembodiment, the percent silk in the solution is greater than 17%. In anembodiment, the percent silk in the solution is greater than 18%. In anembodiment, the percent silk in the solution is greater than 19%. In anembodiment, the percent silk in the solution is greater than 20%. In anembodiment, the percent silk in the solution is greater than 25%. In anembodiment, the percent silk in the solution is between 0.1% and 30%. Inan embodiment, the percent silk in the solution is between 0.1% and 25%.In an embodiment, the percent silk in the solution is between 0.1% and20%. In an embodiment, the percent silk in the solution is between 0.1%and 15%. In an embodiment, the percent silk in the solution is between0.1% and 10%. In an embodiment, the percent silk in the solution isbetween 0.1% and 9%. In an embodiment, the percent silk in the solutionis between 0.1% and 8%. In an embodiment, the percent silk in thesolution is between 0.1% and 7%. In an embodiment, the percent silk inthe solution is between 0.1% and 6.5%. In an embodiment, the percentsilk in the solution is between 0.1% and 6%. In an embodiment, thepercent silk in the solution is between 0.1% and 5.5%. In an embodiment,the percent silk in the solution is between 0.1% and 5%. In anembodiment, the percent silk in the solution is between 0.1% and 4.5%.In an embodiment, the percent silk in the solution is between 0.1% and4%. In an embodiment, the percent silk in the solution is between 0.1%and 3.5%. In an embodiment, the percent silk in the solution is between0.1% and 3%. In an embodiment, the percent silk in the solution isbetween 0.1% and 2.5%. In an embodiment, the percent silk in thesolution is between 0.1% and 2.0%. In an embodiment, the percent silk inthe solution is between 0.1% and 2.4%. In an embodiment, the percentsilk in the solution is between 0.5% and 5%. In an embodiment, thepercent silk in the solution is between 0.5% and 4.5%. In an embodiment,the percent silk in the solution is between 0.5% and 4%. In anembodiment, the percent silk in the solution is between 0.5% and 3.5%.In an embodiment, the percent silk in the solution is between 0.5% and3%. In an embodiment, the percent silk in the solution is between 0.5%and 2.5%. In an embodiment, the percent silk in the solution is between1 and 4%. In an embodiment, the percent silk in the solution is between1 and 3.5%. In an embodiment, the percent silk in the solution isbetween 1 and 3%. In an embodiment, the percent silk in the solution isbetween 1 and 2.5%. In an embodiment, the percent silk in the solutionis between 1 and 2.4%. In an embodiment, the percent silk in thesolution is between 1 and 2%. In an embodiment, the percent silk in thesolution is between 20% and 30%. In an embodiment, the percent silk inthe solution is between 0.1% and 6%. In an embodiment, the percent silkin the solution is between 6% and 10%. In an embodiment, the percentsilk in the solution is between 6% and 8%. In an embodiment, the percentsilk in the solution is between 6% and 9%. In an embodiment, the percentsilk in the solution is between 10% and 20%. In an embodiment, thepercent silk in the solution is between 11% and 19%. In an embodiment,the percent silk in the solution is between 12% and 18%. In anembodiment, the percent silk in the solution is between 13% and 17%. Inan embodiment, the percent silk in the solution is between 14% and 16%.In an embodiment, the percent silk in the solution is 2.4%. In anembodiment, the percent silk in the solution is 2.0%.

In an embodiment, the percent sericin in the solution is non-detectableto 30%. In an embodiment, the percent sericin in the solution isnon-detectable to 5%. In an embodiment, the percent sericin in thesolution is 1%. In an embodiment, the percent sericin in the solution is2%. In an embodiment, the percent sericin in the solution is 3%. In anembodiment, the percent sericin in the solution is 4%. In an embodiment,the percent sericin in the solution is 5%. In an embodiment, the percentsericin in the solution is 10%. In an embodiment, the percent sericin inthe solution is 30%.

In an embodiment, the stability of the LiBr-silk fragment solution is 0to 1 year. In an embodiment, the stability of the LiBr-silk fragmentsolution is 0 to 2 years. In an embodiment, the stability of theLiBr-silk fragment solution is 0 to 3 years. In an embodiment, thestability of the LiBr-silk fragment solution is 0 to 4 years. In anembodiment, the stability of the LiBr-silk fragment solution is 0 to 5years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 1 to 2 years. In an embodiment, the stability of theLiBr-silk fragment solution is 1 to 3 years. In an embodiment, thestability of the LiBr-silk fragment solution is 1 to 4 years. In anembodiment, the stability of the LiBr-silk fragment solution is 1 to 5years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 2 to 3 years. In an embodiment, the stability of theLiBr-silk fragment solution is 2 to 4 years. In an embodiment, thestability of the LiBr-silk fragment solution is 2 to 5 years. In anembodiment, the stability of the LiBr-silk fragment solution is 3 to 4years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 3 to 5 years. In an embodiment, the stability of theLiBr-silk fragment solution is 4 to 5 years.

In an embodiment, the stability of a composition of the presentdisclosure is 10 days to 6 months. In an embodiment, the stability of acomposition of the present disclosure is 6 months to 12 months. In anembodiment, the stability of a composition of the present disclosure is12 months to 18 months. In an embodiment, the stability of a compositionof the present disclosure is 18 months to 24 months. In an embodiment,the stability of a composition of the present disclosure is 24 months to30 months. In an embodiment, the stability of a composition of thepresent disclosure is 30 months to 36 months. In an embodiment, thestability of a composition of the present disclosure is 36 months to 48months. In an embodiment, the stability of a composition of the presentdisclosure is 48 months to 60 months.

In an embodiment, a composition of the present disclosure includes puresilk fibroin-based protein fragments having an average weight averagemolecular weight ranging from 6 kDa to 16 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 17 kDa to 38 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 39 kDa to80 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 1 to 5 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 5 to 10 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 10 to 15 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 15 to 20kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 20 to 25 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 25 to 30 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 30 to 35kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 35 to 40 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 40 to 45 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 45 to 50kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 50 to 55 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 55 to 60 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 60 to 65kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 65 to 70 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 70 to 75 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 75 to 80kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 80 to 85 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 85 to 90 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 90 to 95kDa. In an embodiment, a composition of the present disclosure includespure silk fibroin-based protein fragments having an average weightaverage molecular weight ranging from 95 to 100 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 100 to 105 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 105 to110 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 110 to 115 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 115 to 120 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 120 to 125 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 125 to130 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 130 to 135 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 135 to 140 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 140 to 145 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 145 to150 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 150 to 155 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 155 to 160 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 160 to 165 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 165 to170 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 170 to 175 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 175 to 180 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 180 to 185 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 185 to190 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 190 to 195 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 195 to 200 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 200 to 205 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 205 to210 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 210 to 215 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 215 to 220 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 220 to 225 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 225 to230 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 230 to 235 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 235 to 240 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 240 to 245 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 245 to250 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 250 to 255 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 255 to 260 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 260 to 265 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 265 to270 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 270 to 275 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 275 to 280 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 280 to 285 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 285 to290 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 290 to 295 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 295 to 300 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 300 to 305 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 305 to310 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 310 to 315 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 315 to 320 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 320 to 325 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 325 to330 kDa. In an embodiment, a composition of the present disclosureincludes pure silk fibroin-based protein fragments having an averageweight average molecular weight ranging from 330 to 335 kDa. In anembodiment, a composition of the present disclosure includes pure silkfibroin-based protein fragments having an average weight averagemolecular weight ranging from 35 to 340 kDa. In an embodiment, acomposition of the present disclosure includes pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from 340 to 345 kDa. In an embodiment, a composition of thepresent disclosure includes pure silk fibroin-based protein fragmentshaving an average weight average molecular weight ranging from 345 to350 kDa.

In an embodiment, a composition of the present disclosure having puresilk fibroin-based protein fragments has a polydispersity ranging fromabout 1 to about 5.0. In an embodiment, a composition of the presentdisclosure having pure silk fibroin-based protein fragments has apolydispersity ranging from about 1.5 to about 3.0. In an embodiment, acomposition of the present disclosure having pure silk fibroin-basedprotein fragments has a polydispersity ranging from about 1 to about1.5. In an embodiment, a composition of the present disclosure havingpure silk fibroin-based protein fragments has a polydispersity rangingfrom about 1.5 to about 2.0. In an embodiment, a composition of thepresent disclosure having pure silk fibroin-based protein fragments hasa polydispersity ranging from about 2.0 to about 2.5. In an embodiment,a composition of the present disclosure having pure silk fibroin-basedprotein fragments, has a polydispersity ranging from about is 2.0 toabout 3.0. In an embodiment, a composition of the present disclosurehaving pure silk fibroin-based protein fragments, has a polydispersityranging from about is 2.5 to about 3.0.

In an embodiment, a composition of the present disclosure having puresilk fibroin-based protein fragments has non-detectable levels of LiBrresiduals. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is between 10 ppm and 1000 ppm. Inan embodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is between 10 ppm and 300 ppm. In an embodiment, theamount of the LiBr residuals in a composition of the present disclosureis less than 25 ppm. In an embodiment, the amount of the LiBr residualsin a composition of the present disclosure is less than 50 ppm. In anembodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is less than 75 ppm. In an embodiment, the amount ofthe LiBr residuals in a composition of the present disclosure is lessthan 100 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is less than 200 ppm. In anembodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is less than 300 ppm. In an embodiment, the amount ofthe LiBr residuals in a composition of the present disclosure is lessthan 400 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is less than 500 ppm. In anembodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is less than 600 ppm. In an embodiment, the amount ofthe LiBr residuals in a composition of the present disclosure is lessthan 700 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is less than 800 ppm. In anembodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is less than 900 ppm. In an embodiment, the amount ofthe LiBr residuals in a composition of the present disclosure is lessthan 1000 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is non-detectable to 500 ppm. Inan embodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is non-detectable to 450 ppm. In an embodiment, theamount of the LiBr residuals in a composition of the present disclosureis non-detectable to 400 ppm. In an embodiment, the amount of the LiBrresiduals in a composition of the present disclosure is non-detectableto 350 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is non-detectable to 300 ppm. Inan embodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is non-detectable to 250 ppm. In an embodiment, theamount of the LiBr residuals in a composition of the present disclosureis non-detectable to 200 ppm. In an embodiment, the amount of the LiBrresiduals in a composition of the present disclosure is non-detectableto 150 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is non-detectable to 100 ppm. Inan embodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is 100 ppm to 200 ppm. In an embodiment, the amountof the LiBr residuals in a composition of the present disclosure is 200ppm to 300 ppm. In an embodiment, the amount of the LiBr residuals in acomposition of the present disclosure is 300 ppm to 400 ppm. In anembodiment, the amount of the LiBr residuals in a composition of thepresent disclosure is 400 ppm to 500 ppm.

In an embodiment, a composition of the present disclosure having puresilk fibroin-based protein fragments, has non-detectable levels ofNa₂CO₃ residuals. In an embodiment, the amount of the Na₂CO₃ residualsin a composition of the present disclosure is less than 100 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a composition of thepresent disclosure is less than 200 ppm. In an embodiment, the amount ofthe Na₂CO₃ residuals in a composition of the present disclosure is lessthan 300 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in acomposition of the present disclosure is less than 400 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a composition of thepresent disclosure is less than 500 ppm. In an embodiment, the amount ofthe Na₂CO₃ residuals in a composition of the present disclosure is lessthan 600 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in acomposition of the present disclosure is less than 700 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a composition of thepresent disclosure is less than 800 ppm. In an embodiment, the amount ofthe Na₂CO₃ residuals in a composition of the present disclosure is lessthan 900 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in acomposition of the present disclosure is less than 1000 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a composition of thepresent disclosure is non-detectable to 500 ppm. In an embodiment, theamount of the Na₂CO₃ residuals in a composition of the presentdisclosure is non-detectable to 450 ppm. In an embodiment, the amount ofthe Na₂CO₃ residuals in a composition of the present disclosure isnon-detectable to 400 ppm. In an embodiment, the amount of the Na₂CO₃residuals in a composition of the present disclosure is non-detectableto 350 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in acomposition of the present disclosure is non-detectable to 300 ppm. Inan embodiment, the amount of the Na₂CO₃ residuals in a composition ofthe present disclosure is non-detectable to 250 ppm. In an embodiment,the amount of the Na₂CO₃ residuals in a composition of the presentdisclosure is non-detectable to 200 ppm. In an embodiment, the amount ofthe Na₂CO₃ residuals in a composition of the present disclosure isnon-detectable to 150 ppm. In an embodiment, the amount of the Na₂CO₃residuals in a composition of the present disclosure is non-detectableto 100 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in acomposition of the present disclosure is 100 ppm to 200 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a composition of thepresent disclosure is 200 ppm to 300 ppm. In an embodiment, the amountof the Na₂CO₃ residuals in a composition of the present disclosure is300 ppm to 400 ppm. In an embodiment, the amount of the Na₂CO₃ residualsin a composition of the present disclosure is 400 ppm to 500 ppm.

In an embodiment, the water solubility of pure silk fibroin-basedprotein fragments of the present disclosure is 50 to 100%. In anembodiment, the water solubility of pure silk fibroin-based proteinfragments of the present disclosure is 60 to 100%. In an embodiment, thewater solubility of pure silk fibroin-based protein fragments of thepresent disclosure is 70 to 100%. In an embodiment, the water solubilityof pure silk fibroin-based protein fragments of the present disclosureis 80 to 100%. In an embodiment, the water solubility is 90 to 100%. Inan embodiment, the silk fibroin-based fragments of the presentdisclosure are non-soluble in aqueous solutions.

In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 50 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 60 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 70 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 80 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 90 to 100%.In an embodiment, the silk fibroin-based fragments of the presentdisclosure are non-soluble in organic solutions.

In an embodiment, the percent water content in gels of the presentdisclosure is 20% to 99.9%. In an embodiment, the percent water contentin gels of the present disclosure is 20% to 25%. In an embodiment, thepercent water content in gels of the present disclosure is 25% to 30%.In an embodiment, the percent water content in gels of the presentdisclosure is 30% to 35%. In an embodiment, the percent water content ingels of the present disclosure is 35% to 40%. In an embodiment, thepercent water content in gels of the present disclosure is 40% to 45%.In an embodiment, the percent water content in gels of the presentdisclosure is 45% to 50%. In an embodiment, the percent water content ingels of the present disclosure is 50% to 55%. In an embodiment, thepercent water content in gels of the present disclosure is 55% to 60%.In an embodiment, the percent water content in gels of the presentdisclosure is 60% to 65%. In an embodiment, the percent water in gelcosmetic gels of the present disclosure s is 65% to 70%. In anembodiment, the percent water content in gels of the present disclosureis 70% to 75%. In an embodiment, the percent water content in gels ofthe present disclosure is 75% to 80%. In an embodiment, the percentwater content in gels of the present disclosure is 80% to 85%. In anembodiment, the percent water content in gels of the present disclosureis 85% to 90%. In an embodiment, the percent water content in gels ofthe present disclosure is 90% to 95%. In an embodiment, the percentwater content in gels of the present disclosure is 95% to 99%.

In an embodiment, the percent water content in films of the presentdisclosure is 20%. In an embodiment, the percent water content in filmsof the present disclosure is less than 20%. In an embodiment, thepercent water content in films of the present disclosure is less than18%. In an embodiment, the percent water content in films of the presentdisclosure is less than 16%. In an embodiment, the percent water contentin films of the present disclosure is less than 14%. In an embodiment,the percent water content in films of the present disclosure is lessthan 12%. In an embodiment, the percent water content in films of thepresent disclosure is less than 10%. In an embodiment, the percent watercontent in films of the present disclosure is between about 2% and about20%.

In an embodiment, the extraction temperature during a method ofpreparing a composition of the present disclosure is greater than 84° C.In an embodiment, the extraction temperature during a method ofpreparing a composition of the present disclosure is less than 100° C.In an embodiment, the extraction temperature during a method ofpreparing a composition of the present disclosure is 84° C. to 100° C.In an embodiment, the extraction temperature during a method ofpreparing a composition of the present disclosure is 84° C. to 94° C. Inan embodiment, the extraction temperature during a method of preparing acomposition of the present disclosure is 94° C. to 100° C.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described embodiments, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLES Example 1 Development of a Silk Film of the Present Disclosurefor use in Fine Line Lifting Applications

TABLE 18 Film Recipe for Fine Line Lifting Film - FIG. 82A % SPF MixtureSolution of the 2.4% Present Disclosure Quantity Vitamin C 4:1 (silk:VitC) (0.006 g/mL 2.4% solution) 20% mL per film (2.5 cm by 10 cm) 7.08 mLMass of silk per film:  170 mg Mass of l-ascorbic acid per film: 42.5 mgpH 4.0 (when water is applied)

Silk films (2.5 cm×10 cm) were manufactured according to methodsdisclosed herein varying process parameters so as to result in fine linelifting films. The silk films were given the name “PureProC™ film”, andcan be packaged in a foil based package that is air tight and lightproof. Table 18 provides details of the PureProC™ films used in a studyof 32 individuals using the films for four (4) weeks. Biocompatibilityand hypo-allergenicity of the films was observed. Further, nosensitization, toxicity, or immune response was observed. FIG. 84 is agraph summarizing the quantity of vitamin C in a daily dose (i.e., theaverage amount of product used to cover a 25 cm² area of skin) ofPureProC™ and competitor products over a 30 day period. FIGS. 85 and 86summarize resultant ease of use data and observed benefits within thefirst month of use.

In an embodiment, PureProC™ films were removed by peeling the films off.In an embodiment, PureProC™ films were removed by using a wet cottonball or similar removal pad. In an embodiment, PureProC™ films wereremoved by washing the area where the film is placed with a wash cloth.In an embodiment, PureProC™ film PureProC™ films were removed usingwater. The PureProC™ films can be shaped into strips for multiple areasof the face or larger pieces can be cut to fit target areas. In anembodiment, grips or backing(s) on the PureProC™ films can be includedfor ease of application. In an embodiment, a PureProC™ film of thepresent disclosure includes silk and vitamin C (20%).

In an embodiment, a film of the present disclosure is soluble in water(insoluble border). In an embodiment, a film of the present disclosureis clear/transparent. In an embodiment, a film of the present disclosurehas a pH=4 when water is applied. Films of the present disclosure can bemade with different combinations of % silk and volume to produce filmswith silk quantities of 3 mg/cm̂2 to 10 mg/cm̂2. Films of the presentdisclosure can be made with from about 1% to about 50% 1-ascorbic acid.Films of the present disclosure can adhere to skin with water. Films ofthe present disclosure can be spread on skin once water is applied.Films of the present disclosure can dry when humidity of dryingequipment is 16-40% and below the humidity of the lab

Example 2 Development of Silk Gels of the Present Disclosure

TABLE 19 Gel Samples - Silk gel formulations including additives,concentration of silk and additive, gelation conditions and gelationtimes. mL 2% Mass Amount Sample silk Vit C Ratio of Temp/ Days to Namesolution (g) silk:VitC Additive additive Treatment Gelation 1 10 0.045:01 None None RT 8 2 10 0.08 2.5:1   None None RT 8 3 10 0.2 1:01 NoneNone RT 8 4 10 0.4 1:02 None None RT 14 5 10 0.8 1:04 None None RT None6 10 0.04 5:01 None None Fridge ~39 7 10 0.08 2.5:1   None None Fridge~39 8 10 0.2 1:01 None None Fridge ~39 9 10 0.4 1:02 None None FridgeNone 10  10 0.8 1:04 None None Fridge None 11  10 0.2 1:01 None NoneRT/Shake 8 vigorously O-1 10 0.04 5:01 None None 37 C. Oven 3 O-2 100.04 5:01 None None 50 C. Oven 2 O-3 10 0.2 1:01 None None 37 C. Oven 4O-4 10 0.2 1:01 None None 50 C. Oven 3 M 40 0.16 5:01 None None RT 5 D40 0.16 5:01 None None RT 5 E1 10 0.04 5:01 Vit E 1 drop RT 7 E2 10 0.045:01 Vit E 3 drops RT 7 E3 10 0 None Vit E 1 drop RT None E4 10 0 NoneVit E 3 drops RT None L1 10 0.04 5:01 Lemon  300 uL RT 6 L2 10 0.04 5:01Lemon Juice  300 uL RT 6 L3 10 0.04 5:01 Lemon Juice 1000 uL RT 5 L4 100 None Lemon  300 uL RT 6 L5 10 0 None Lemon Juice  300 uL RT 7 Jar 1 200.08 5:01 Lemon Juice 2000 uL RT 5-7 Jar 2 5 0.02 5:01 Lemongrass 1 dropRT 2-3 Oil R-1 10 0.04 5:01 Rosemary 1 drop RT 7 Oil T-1 10 0.04 5:01None None RT/Tube 7 RO-1 10 0.04 5:01 Rose Oil 1 drop RT 6 RO-2 10 NoneNone Rose Oil 1 drop RT None

Ratio of Silk to Vitamin C

Samples 1-10 were used to examine the effect of silk to vitamin C ratioon serum gelation. Samples 1-3 with less vitamin C gelled quicker thansamples 4 and 5. All other conditions were kept constant. Samples 6-8with less vitamin C gelled quicker than samples 9 and 10. All otherconditions were kept constant. It is concluded that decreasing the ratioof silk to vitamin C (increasing the amount of vitamin C), will lengthenthe time to gel creation. At ratios with small amounts of vitamin C,days to gel creation did not vary greatly.

Physical Stimulation

Samples 3 and 11 were used to examine the effect of physical stimulationon serum gelation. Each sample was prepared under the same conditions.Sample 11 was vigorously shaken for about 3 minutes after addition ofvitamin C. Treatment of 3 and 11 was otherwise the same. The shakingresulted in bubbles but did not significantly change gel creation time.

Temperature Treatment

Samples 1, 3, 6, 8, O-1, O-2, O-3, and O-4 were used to examine theeffect of temperature treatment on serum gelation time. Samples 1, 6,O-1, and O-2 were identical other than temperature treatment. Samples 3,8, O-3, and O-4 were identical other than temperature treatment. The twogroups differed in silk to vitamin C ratio. Time to serum gelation wasdirectly related to temperature treatment with a higher temperatureresulting in quicker serum gelation.

Solution Volume

Samples 1, M and D were used to examine the effect of solution volume onserum gelation time. Samples M and D varied from sample 1 only by anincreased solution volume. Samples M and D gelled in 5 days while sample1 gelled in 8 days. Samples M and D were definitively noticed to begelled on the day of gelling while sample 1 gelled over a weekend.

Additives

Samples E1, E2, E3, E4, L1, L2, L3, L4, L5, Jar 2, R1, RO-1 and RO-2were used to examine the effect of additives on serum gelation time.Samples E1-4 contained Vitamin E. Only samples E1 and E2 containedvitamin C and only these two samples gelled. Vitamin E can be added to asolution to become a gel but it appears that another additive may beneeded to create a gel. Samples L1-5 contained a form of lemon juice.Samples L1 and L4 had juice directly from a lemon while samples L2, L3and L5 contained lemon juice from a plastic lemon container. Samples L4and L5 did not have vitamin C while all others did. All samples gelledshowing that lemon juice can create gel on its own. Amount of lemonjuice and type of lemon juice had little effect on gelation time. SampleJar 2 contained lemon grass oil which formed an albumen like substancewhen initially added. This sample also had vitamin C but gelation timewas significantly quicker than with other vitamin C samples. Sample R1contained rosemary oil, which seemed to be soluble, as well as vitaminC. The sample gelled in a similar time frame to other samples with onlyvitamin C. Samples RO-1 and RO-2 contained rose oil while only RO-1 hadvitamin C. Only RO-1 gelled showing that rose oil will not create a gelquickly on its own. In both cases the rose oil was immiscible andvisible as yellow bubbles.

Aqueous silk fibroin-based fragment solution and essential oils areimmiscible liquids. In an embodiment, to increase the fragrance of thesilk fibroin-based fragment solution, without entrapping oils within thesolution, the solution is mixed with the essential oil with the use of astir bar. The stir bar is rotated at a speed such that some turbulenceis observed in the mixture, thus causing contact between the fragrantessential oil and the molecules in solution, adding a scent to thesolution. Before casting of product from the solution, mixing may bestopped and the oil allowed to separate to the top of the solution.Dispensing from the bottom fraction of the solution into the finalproduct allows for fragrance without visible essential oil within thefinal product.

Alternatively, the silk fibroin-based solution and essential oil can becombined with or without additional ingredients and/or an emulsifier tocreate a composition containing both ingredients.

In an embodiment, mixing of the solution as described above can reducegelation time if the solution is used to create a gel formulation.

Vessel

Samples T1 and Jar 1 were used to examine the effect of casting vesselon serum gelation time. Jar 1 was cast in a glass jar while T1 was castin an aluminum tube. Both samples gelled and did not affect serum geltime.

Summary

All treatments of silk solution for gel solution were in a conical tubeat room temperature unless otherwise stated. The ratio of silk tovitamin C did affect the ability of a solution to gel as ratios above1:2 did not gel and a 1:2 ratio took twice as long as other lower ratios(5:1, 2.5:1, 1:1). Temperature affected gel creation time with highertemperatures resulting in quicker gel times. 50° C. treatment gelled inas quick as 2 days, 37° C. treatment gelled in as quick as 3 days, roomtemperature treatment gelled in 5-8 days and storage in a refrigeratortook at least 39 days to gel. The effects of additives on gel creationwere dependent on the additive. Vitamin E, Rosemary Oil and Rose Oil allhad no effect on gel creation. Each of these additives did not preventgelation or affect the time to gelation. Each also required the presenceof vitamin C to gel. Lemon juice from a fresh lemon, pre-squeezed lemonjuice from a plastic lemon container and lemon grass oil did affect gelcreation. Without wishing to be bound by theory, it is believed that thelower pH as a result of these additives is the reason the additives hadan impact on decreasing gelation time. Both lemon juice types were ableto cause gelation without the presence of vitamin C. This occurred inthe same number of days as with vitamin C. The lemongrass oil was ableto decrease the number of days to gelation to 2-3 days. All additivesappeared soluble other than lemongrass oil and rose oil. Rose oilremained in yellow bubbles while the lemongrass oil was partiallysoluble and formed an albumen like chunk. In an embodiment, oils thatare not fully soluble, can still be suspended within the gel as anadditive. Physical stimulation by shaking, vessel the solution was castinto and solution volume did not affect gelation time. FIG. 81 is agraph representing the % Activity of Vitamin C in gels of the presentdisclosure.

TABLE 20 Concentration of vitamin C in various gel formulations. SampleConcentration of Weight Vitamin C (mg/g) Sample Info (mg) In SampleAverage Rosemary 685.7 3.2511 3.2657 (Room 3.2804 Temperature 638 3.33363.3334 storage) 3.3332 Lemongrass 646 2.8672 2.877 (Room 2.8868Temperature 645.5 2.9051 2.9051 storage) 2.9052 Rosemary 645.2 3.90633.9147 (Room 3.923 Temperature; 649 3.9443 3.9374 Foil Covered 3.9305storage) Lemongrass 630.1 3.8253 3.8274 (Room 3.8295 Temperature; 660.43.8283 3.8253 Foil Covered 3.8222 storage) Rosemary 672.4 5.1616 5.1484(Fridge, Foil 5.1352 Covered 616.5 5.1984 5.201 storage) 5.2036Lemongrass 640.5 5.1871 5.1824 (Fridge, Foil 5.1776 Covered 627.7 5.20985.2126 storage) 5.2154

Example 3 Development of Silk Gels of the Present Disclosure for use asSmoothing Gel

TABLE 21 Lemongrass Gel % Silk Solution 2% Quantity Vitamin C 100 mg/15mL solution Quantity Lemongrass Oil 20 uL/15 mL solution

TABLE 22 Rosemary Gel % Silk Solution 2% Quantity Vitamin C 100 mg/15 mLsolution Quantity Rosemary Oil 20 uL/50 mL solution

TABLE 23 Lemongrass Gel (50 mL) % Silk Solution (60 minute boil, 25 kDA)2% Quantity Vitamin C (ascorbyl glucoside) 12.82 mg/mL solution (641 mgtotal) Quantity Lemongrass Oil 1.33 uL/mL solution pH 4

TABLE 24 Rosemary Gel (50 mL) % Silk Solution (60 minute boil, 25 kDA)2% Quantity Vitamin C (ascorbyl glucoside) 12.82 mg/mL solution (641 mgtotal) Quantity Rosemary Oil 0.8 uL/mL solution pH 4

Gels of the present disclosure can be made with about 0.5% to about 8%silk solutions. Gels of the present disclosure can be made with ascorbylglucoside at concentrations of about 0.67% to about 15% w/v. Gels of thepresent disclosure be clear/white in color. Gels of the presentdisclosure can have a consistency that is easily spread and absorbed bythe skin. Gels of the present disclosure can produce no visual residueor oily feel after application. Gels of the present disclosure do notbrown over time.

Silk gels with essential oils were prepared by diluting a silk solutionof the present disclosure to 2%. Vitamin C was added to the solution andallowed to dissolve. The essential oil was added, stirred and dissolved.The solution was aliquot into jars.

A trial was conducted with 44 people on two formulations of the presentdisclosure, PureProC™ Rosemary Gel and PureProC™ Lemongrass Gel (FIGS.87 and 88). Respondents were asked to use each sample once a day for aweek each. The majority of respondents applied the gel to the wholeface. Other areas where it was most commonly applied included theforehead, under eyes and near mouth.

The majority of respondents applied the gel during the morning (67%)with the balance 33% applying the gel in the evening. Ninety-eight (98%)of participants used the gel once a day during the test. Respondentswere asked to describe in their own words how the gel felt when it wasapplied and how it felt during the 24 hours until the next application.Smooth, cool, and soft were the most often mentioned adjectives used todescribe how the gel felt. Eighty percent (80%) of test participantsgave a high score to interest in continuing to use the gel.

Respondents were asked about what they did with their other productsthat were usually used on their face during the trial. The majorityapplied the gel first and then added the other products or applied thegel at night with no additional products. Only 14% of participantsindicated that they eliminated one of their normal products whiletesting the gel. PureProC™ can be used in conjunction with or inreplacement of other products. Additionally, sunscreen can be added tothe gel or it may be dispensed from a pump instead of a jar. Withrepeated topical use, no skin irritation, rash, or signs ofnon-compatibility was observed. Biocompatibility and hypo-allergenicityof the gels was observed. Further, no sensitization, toxicity, or immuneresponse was observed.

Example 4 Silk Articles of the Present Disclosure Made From SilkSolutions of the Present Disclosure

Silk solutions of various molecular weights and/or combinations ofmolecular weights can be optimized for specific applications. Thefollowing provides an example of this process but it not intended to belimiting in application or formulation.

Three (3) silk solutions were utilized in standard silk structures inaccordance with standard methods in the literature with the followingresults:

-   -   Solution #1 is a silk concentration of 5.9%, average MW of 19.8        kDa and 2.2 PD (made with a 60 min boil extraction, 100 degree        LiBr dissolution for 1 hr)    -   Solution #2 is a silk concentration of 6.4% (made with a 30 min        boil extraction, 60 degree LiBr dissolution for 4 hrs)    -   Solution #3 is a silk concentration of 6.17% (made with a 30 min        boil extraction, 100 C LiBr dissolution for 1 hour)

Films:

Films were made in accordance with Rockwood et al (Nature Protocols;Vol. 6; No. 10; published on-line Sep. 22, 2011;doi:10.1038/nprot.2011.379). Briefly, 4 mL of 1% or 2% (wt/vol) aqueoussilk solution was added into 100 mm Petri dish (Volume of silk can bevaried for thicker or thinner films and is not critical) and allowed todry overnight uncovered. The bottom of a vacuum desiccator was filledwith water. Dry films were placed in the desiccator and vacuum applied,allowing the films to water anneal for 4 hours prior to removal from thedish. Films cast from solution #1 did not result in a structurallycontinuous film; the film was cracked in several pieces. These pieces offilm dissolved in water in spite of the water annealing treatment.

Egel:

“Egel” is an electrogelation process as described in Rockwood of al.Briefly, 10 ml of aqueous silk solution is added to a 50 ml conical tubeand a pair of platinum wire electrodes immersed into the silk solution.A 20 volt potential was applied to the platinum electrodes for 5minutes, the power supply turned off and the gel collected. Solution #1did not form an EGEL over the 5 minutes of applied electric current.

Gelation:

Solutions #2 and #3 were gelled in accordance with the publishedhorseradish peroxidase (HRP) protocol. Behavior seemed typical ofpublished solutions.

Sonicated Gels

Gels were made following the sonication process in Rockwood et al.Briefly, 5 ml of silk solution was added to a 15 ml conical tube. Thesonicating horn was immersed in the solution and the solution sonicatedat 50% amplitude (21 W). Silk gels were made with 2%, 4% and 6% silksolutions. As compared to standard literature silk, Solutions #2 and #3formed gels after a longer time, for example:

-   -   Standard literature silk: 5-8 min    -   Solution #2: 20 min    -   Solution #3: 120 min        Porous 3D scaffolds:

Water based, salt leached scaffolds were made in accordance with thepublished methods of Rockwood. Salt with particle sizes of interest wasprepared by stacking the sieves with the largest mesh on top and thesmallest mesh on the bottom. Salt was added and sieves shaken vigorouslycollecting the salt. With a 5-ml syringe, 6% (wt/vol) fibroin solutionwas aliquoted into plastic containers, 2 ml per mold and 5-600 micronsalt particles were slowly added on top of the fibroin solution in themold while rotating the container so that the salt was uniform. Theratio of salt to silk in solution was maintained at 25:1.

The container was gently tapped on the bench top to remove air bubbles,the cap closed and the solution allowed to settle overnight at roomtemperature. Once gelled, the lids were removed and the molds placed ina 2-liter beaker with ultrapure water (three containers per 2 liters ofwater). The beakers were transferred to a stir plate and stirred,changing the water 2-3 times per day for 2 d (4-6 washes in total). Thescaffolds were removed from the molds and placed them in fresh water foran additional day.Solution #1 did not form a scaffold; it did not gel. Both solution #2 &#3 formed scaffolds. The scaffolds made with Solution #3 appear softerthan the ones made with Solution #2, but both scaffolds werehomogeneous.

Example 5 Tangential Flow Filtration (TFF) to Remove Solvent fromDissolved Silk Solutions of the Present Disclosure

A variety of % silk concentrations have been produced through the use ofTangential Flow Filtration (TFF). In all cases a 1% silk solution wasused as the input feed. A range of 750-18,000 mL of 1% silk solution wasused as the starting volume. Solution is diafiltered in the TFF toremove lithium bromide. Once below a specified level of residual LiBr,solution undergoes ultrafiltration to increase the concentration throughremoval of water. See examples below.

7.30% Silk Solution:

A 7.30% silk solution was produced beginning with 30 minute extractionbatches of 100 g silk cocoons per batch. Extracted silk fibers were thendissolved using 100 C 9.3M LiBr in a 100 C oven for 1 hour. 100 g ofsilk fibers were dissolved per batch to create 20% silk in LiBr.Dissolved silk in LiBr was then diluted to 1% silk and filtered througha 5 um filter to remove large debris. 15,500 mL of 1%, filtered silksolution was used as the starting volume/diafiltration volume for TFF.Once LiBr was removed, the solution was ultrafiltered to a volume around1300 mL. 1262 mL of 7.30% silk was then collected. Water was added tothe feed to help remove the remaining solution and 547 mL of 3.91% silkwas then collected.

6.44% Silk Solution:

A 6.44% silk solution was produced beginning with 60 minute extractionbatches of a mix of 25, 33, 50, 75 and 100 g silk cocoons per batch.Extracted silk fibers were then dissolved using 100 C 9.3M LiBr in a 100C oven for 1 hour. 35, 42, 50 and 71 g per batch of silk fibers weredissolved to create 20% silk in LiBr and combined. Dissolved silk inLiBr was then diluted to 1% silk and filtered through a 5 um filter toremove large debris. 17,000 mL of 1%, filtered silk solution was used asthe starting volume/diafiltration volume for TFF. Once LiBr was removed,the solution was ultrafiltered to a volume around 3000 mL. 1490 mL of6.44% silk was then collected. Water was added to the feed to helpremove the remaining solution and 1454 mL of 4.88% silk was thencollected

2.70% Silk Solution:

A 2.70% silk solution was produced beginning with 60 minute extractionbatches of 25 g silk cocoons per batch. Extracted silk fibers were thendissolved using 100 C 9.3M LiBr in a 100 C oven for 1 hour. 35.48 g ofsilk fibers were dissolved per batch to create 20% silk in LiBr.Dissolved silk in LiBr was then diluted to 1% silk and filtered througha 5 um filter to remove large debris. 1000 mL of 1%, filtered silksolution was used as the starting volume/diafiltration volume for TFF.Once LiBr was removed, the solution was ultrafiltered to a volume around300 mL. 312 mL of 2.7% silk was then collected.

Example 6 Gel Vitamin C Derivatives of the Present Disclosure

The purest form of vitamin C is L-ascorbic acid. There are a number ofother derivatives of vitamin C that function like pure vitamin C afterthey are converted to L-ascorbic acid in the body. Vitamin C derivativesare being utilized to extend shelf life. Derivatives are stable forms ofL-ascorbic acid and will not oxidize or lose stability. Table 25 belowsummarizes some vitamin C derivatives tested in the skin care productsof the present disclosure:

TABLE 25 Derivatives Explored Sodium Ascorbyl Phosphate (Aromantic)Sodium Ascorbyl Phosphate (DSM) Magnesium Ascorbyl Phosphate AscorbicAcid-2-Glucoside Ascorbyl Tetraisopalmitate

The Tables in FIGS. 89A-89B summarize embodiments of gels of the presentdisclosure. Ascorbic acid-2-glucoside was the most successful vitamin Cderivative at gel formation with gel being formed in a 2% silk solutionin 3 days. Sodium ascorbyl phosphate from DSM supplier formed a gel in a2% silk solution after 28 days while the same molecule from Aromanticfailed to create a gel. In all cases 100 mg of vitamin C derivative wasmixed in 15 mL of 2% silk solution, and all gels had the same appearanceas gels created with ascorbic acid.

Gels were also cast with combinations of two vitamin C options. In eachcase, at least one of the vitamin C options was known to cause gelation(L-ascorbic acid or ascorbic acid-2-glucoside). All combination gelswere able to gel at 1% total vitamin C additive concentration. A gelcast at 20% total vitamin C additive concentration did not gel. Withoutwishing to be bound by theory, it appears there is a relationshipbetween vitamin C concentration, silk concentration, and gelation. Anincrease in vitamin C at a given concentration of silk will result in alonger time to gelation or inhibit gelation. This may be due to thevitamin C molecule physically blocking interaction between silk proteinfragments or cross-linking of silk protein. Modification to pH may allowadditional concentrations of vitamin C and derivatives thereof to beadded.

Ascorbyl tetraisopalmitate was not used in any gel forming formulation,as it was unable to dissolve or be dispersed in an aqueous silksolution. Ascorbyl tetraisopalmitate is a highly viscous, oil solubleliquid that might need the help of an emulsifier to possible dissolve inaqueous silk solution.

Example 7 Film Vitamin C Derivatives of the Present Disclosure

FIG. 90 is a table summarizing embodiments of films of the presentdisclosure. Sodium ascorbyl phosphate, magnesium ascorbyl phosphate andascorbic acid-2-glucoside could be cast in films with varyingappearance. Sodium ascorbyl phosphate films were opaque and white with atextured top surface similar to plastic. Magnesium ascorbyl phosphatefilms were clear and cloudy with a textured top surface similar toplastic. Ascorbic acid-2-glucoside films were most similar to L-ascorbicacid films although slightly less pliable and slightly textured. Allfilms were soluble with an insoluble border. In an embodiment, a filmwith an insoluble border can be made completely spreadable by punching ashape from the region contained within the soluble section.

Example 8 Caffeine Films with Vitamin C of the Present Disclosure

FIGS. 91A-91B are tables summarizing embodiments of caffeine films ofthe present disclosure. Films were cast with 0.5%, 1%, 2.5%, 5%, 10%,15% and 20% caffeine and 20% or 25% vitamin C. All combinations formedfilms. 20% caffeine films had caffeine precipitate out. Films with0.5%-2.5% were soluble. In an embodiment, a caffeine film of the presentdisclosure is used for reducing puffy eyes.

Example 9 Caffeine Gels with Vitamin C of the Present Disclosure

A silk gel with 2% silk and 100 mg L-ascorbic acid/15 mL solution wascreated with the addition of 50 mg caffeine/15 mL solution. The gel hasthe exact appearance of standard L-ascorbic acid gels. In an embodiment,a caffeine gel of the present disclosure is used for reducing puffyeyes. A range of essential oils can be used including, but not limitedto, lemongrass, vanilla, geranium, and green tea.

Example 10 Green Tea Gels with Vitamin C of the Present Disclosure

Steps:

Green Tea Prep Heat 250 mL water to boil Steep tea bag 2-3 minutes withoccasional stir remove tea bag and let cool Gel Solution Use TFF-10-0047(3.71% silk) Prep dilute to 3% silk with water dilute to 2% with greentea add L-ascorbic acid Gel Gelation occurred like standard gel at roomtemperature Green/yellow color Green Tea scent Solution Spec: 2% silksolution 65 mL (35 ml of 3.71% silk, 8.3 mL water, 21.66 mL green tea)0.43 g L-ascorbic acid

FIG. 92 is a table summarizing an embodiment of a caffeine gel of thepresent disclosure. A silk gel with 2% silk and 100 mg L-ascorbicacid/15 mL solution was created with the addition of 50 mg caffeine/15mL solution. The gel has the exact appearance of standard L-ascorbicacid gels.

Example 11 Preservative Gels with Vitamin C of the Present Disclosure

FIG. 93 is a table summarizing embodiments of preservative gels of thepresent disclosure. Silk gels were cast with standard 2% silk solutionand 100 mg L-ascorbic acid/15 mL solution with the addition of apreservative and chelating agent. The preservative added was VerstatilSL by Kinetic (Water, Sodium Levulinate, Potassium Sorbate) at 1.5% andthe chelating agent was Dermofeel-PA3 by Kinetic (Sodium Phytate) at0.1%. The addition of preservatives extended gelation time to 7 days.Gel is being observed for discoloration and integrity with L-ascorbicacid and ascorbic acid-2-glucoside gel comparisons.

Example 12 Chemical Peels of the Present Disclosure

The primary variable investigated was the concentration of lactic acidand/or glycolic acid necessary to create a silk solution of a desiredpH. In order to determine the relationship between concentration in silkand pH, 2% silk solutions (60 minute boil, 25 kDA) were titrated withglycolic and lactic acid and tested for pH with pH strips. See thefollowing titrations/formulations below:

TABLE 26 Lactic Acid Peel 1: Initial solution: 25 mL of 2% silksolution, pH = 7-8 Quantity of Lactic Acid Added Total Lactic Acid pH100 μL 100 μL 3 100 μL 200 μL 2 100 μL 300 μL 1-2 Time to gel: 3 days

TABLE 27 Lactic Acid Peel 2: Initial solution: 25 mL of 2% silksolution, pH = 7-8 Quantity of Lactic Acid Added Total Lactic Acid pH 25μL 25 μL 4 Time to gel: >5 days

TABLE 28 Glycolic Acid Peel 1: Initial solution: 25 mL of 2% silksolution, pH = 7-8 Quantity of Glycolic Acid Added Total Glycolic AcidpH   41 mg    41 mg 4 43.25 mg  84.25 mg 3  30.7 mg 114.95 mg 3  56.4 mg171.35 mg 2-3 91.66 mg 263.01 mg 2 171.35 mg   434.4 mg 1-2 Time to gel:3 days

TABLE 29 Glycolic Acid Peel 2: Initial solution: 25 mL of 2% silksolution, pH = 7-8 Quantity of Lactic Acid Added Total Lactic Acid pH 41mg 41 mg 4 Time to gel: >5 days

TABLE 30 Lactic/Glycolic Acid Peel: Initial solution: 25 mL of 2% silksolution, pH = 7-8 Total Total Glycolic Lactic Acid Acid Lemongrass pH150 μL 200 mg 33.3 μL 2 Time to gel: 3 days

TABLE 31 Lactic/Glycolic Acid Peel: Initial solution: 30 mL of 2% silksolution, pH = 7-8 % Silk Solution (60 minute boil, 25 kDA) 2% LacticAcid Concentration 6 μL/mL Glycolic Acid Concentration 8 mg/mL pH 2Lemongrass Concentration 1.33 μL/mL

A peel of the present disclosure can have a % silk ranging from about0.5% to about 8%. The pH of a peel of the present disclosure can beadjusted with varying quantities of lactic and glycolic acid. Peels canalso be made with lactic acid only or glycolic acid only. A peel of thepresent disclosure can be clear/white in color. A peel of the presentdisclosure can have a gel consistency that is easily spread and absorbedby the skin. A peel of the present disclosure does not brown or changecolors.

In an embodiment, a chemical peel of the present disclosure can beapplied weekly to reveal healthy, vibrant skin. In an embodiment, achemical peel of the present disclosure can be applied weekly todiminish fine lines. In an embodiment, a chemical peel of the presentdisclosure can be applied weekly to firm the skin.

Each formulation (after titration, if applicable) was applied as aliquid and as a gel and observed for look and feel. Peels of pH=4(Lactic Acid Peel 2, Glycolic Acid peel 2) resulted in a minimal burningfeeling after a few minutes of application, while peels of pH=˜2 (LacticAcid Peel 1, Glycolic Acid Peel 1, Lactic/Glycolic Acid Peel) caused aslightly more intense burning feel. Little difference in degree ofburning was felt between liquid and gel other than that the burningsensation was more delayed in the gel form. PH was maintained in the gelform and was confirmed by using a pH strip.

Glycolic acid and lactic acid are both alpha hydroxy acids (AHA's) thatare among the most commonly used peels for superficial peeling(outermost skin layer peeling). Chemical peels are intended to burn thetop layers of the skin in a controlled manner, to remove superficialdermal layers and dead skin in order to improve appearance. AHAs arecommon in chemical peels due to low risk of adverse reactions and highcontrol of strength (control pH and time applied). Glycolic acid is mostcommonly used and has a very small molecular size, enabling deeppenetration into the epidermis. Lactic acid is another commonly used AHAand offers a more gentle peel with higher control due to its largermolecular size. Any number of chemicals known in the art that lower pHand are physical exfoliates can be used in place of AHAs.

Example 13 Hydrating Serums of the Present Disclosure

Variables include: concentration of silk in solution, concentration ofHA, addition of vitamin C, and serum preparation method. Table 32 is alist of samples that were evaluated:

TABLE 32 Embodiments of serums of the present disclosure containing HAand Silk (60 minute boil, 25 kDA), with or without vitamin C, and with20 uL/15 mL lemongrass essential oil (30 mL solution) Silk Vit C MethodHA (%) (%) (mg) Observation HA added to water 0.5 2 0 White, slightlyopaque, viscous liquid before dilution of 1 White/yellow, slightlyopaque, viscous liquid silk 0.5 2 0 Low viscosity, clear-white opaquewith film on top, some white residue when applied topically to skin 1Slightly viscous, clear liquid with film on top 0.5 1 0 Slightlyviscous, clear liquid with film on top 1 Smooth viscous liquid, no whiteresidue when applied topically to skin 0.5 0.5 0 Moderately viscousliquid, clear 1 Smooth, clear, no white residue when applied topicallyto skin 0.5 2 35 Non homogeneous mix of hard gel and viscous liquid 1Non homogeneous mix of hard gel and viscous liquid 1 1 35 Nonhomogeneous mix of hard gel and viscous liquid 0.5 Opaque, whiteliquid/non-viscous 1 4 35 Separated mixture of hard gel and viscousliquid 0 Non homogeneous mix of hard gel and viscous liquid 5 2 0Yellow, gel HA added to water 10 2 0 Viscous jelly upon stirring withundissolved HA before dilution of 5 Very viscous jelly upon stirringsilk, stirred 1 Viscous jelly upon stirring vigorously 0.5 HA added towater 1 2 0 Non homogeneous thick, viscous jelly/gel before dilution of5 1 0 silk, shaken HA added to water 1 1 0 Clear/slightly opaque,viscous liquid, smooth and let sit for 1 day feel, little to no whiteresidue when applied before dilution of topically to skin silk HA addedto 0.5 2 0 Viscous, clear/white liquid varying in diluted silk 1consistency solution, stirred 0.5 1 Clear viscous liquid varying inconsistency 1 0.5 6 White, opaque jelly varying in consistency 1 HAadded to 0.5 3.9 0 White, slightly opaque, viscous liquid diluted silk 1solution, stirred 0.5 2 35 White gel varying in consistency 1

In an embodiment, a hydrating serum of the present disclosure protectsthe skin and seals in moisture with the power of silk fibroin-basedfragment proteins. In an embodiment, a hydrating serum of the presentdisclosure delivers moisture for immediate and long-term hydrationthroughout the day with concentrated hyaluronic acid. A range ofessential oils can be used in a hydrating serum of the presentdisclosure including, but not limited to, lemongrass, vanilla, geranium,and green tea. In an embodiment, one or two drops of a hydrating serumof the present disclosure can be smoothed over the face and neck. In anembodiment, a hydrating serum of the present disclosure includes water,aqueous silk fibroin-based fragment solution, hyaluronic acid, andlemongrass oil. In an embodiment, the silk fibroin-based fragmentprotein in a hydrating serum of the present disclosure has the abilityto stabilize and protect skin while sealing in moisture, all without theuse of harsh chemical preservatives or synthetic additives. In anembodiment, the hyaluronic acid in a hydrating serum of the presentdisclosure nourishes skin and delivers moisture for lasting hydration.In an embodiment, the lemongrass essential oil in a hydrating serum ofthe present disclosure yields antioxidant and anti-inflammatoryproperties that support skin rejuvenation. In an embodiment, a hydratingserum of the present disclosure has a pH of about 6.0.

Silk Fibroin-Based Fragment Solution

Because silk fibroin-based fragment solution is both aqueous and able toentrap and deliver small molecules, the solution is able to deliver bothwater and hygroscopic HA molecules to the skin for hydration. A range inconcentration of silk fibroin-based fragment compositions in solutionfrom 0.5%-6.0% was tested for feasibility and product outcome. Allconcentrations tested were found to be feasible.

Hyaluronic Acid

Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in thehydrating serum due to its hygroscopic properties and ability to promotesoft, hydrated skin. A range in concentration of hyaluronic acid insolution from 0.5%-10.0% was tested for feasibility and product outcome.All concentrations tested, with the exception of 10.0%, were found to befeasible. Feasibility was determined based on the ability to dissolvehyaluronic acid.

Vitamin C and Derivatives Thereof

Vitamin C (L-ascorbic acid) was tested as an ingredient in the hydratingserum. Initial vitamin C samples became a non-homogeneous mixture of geland liquid. A follow-up trial with vitamin C resulted in a homogeneous,white, opaque, non-viscous liquid that was not quickly absorbed by theskin. In an embodiment, a vitamin C derivative that does not readilycause gelation, such as sodium ascorbyl phosphate, could be added up tothe concentration at which it would no longer be soluble (for example,0% to about 40%). In an embodiment, 20% sodium ascorbyl phosphate couldbe added. Vitamin C options that do cause gelation (L-ascorbic acid andascorbyl glucoside) could be added at high concentrations (for examplegreater than about 10% up to about 50%) at which gelation is inhibited.

Serum Creation Method

Initial serums were created by the addition of HA to a silkfibroin-based fragment solution followed by stirring. The HA appeared tostick together and was not dissolved until forcefully stirred. Themixing process was then changed so that HA was first dissolved in waterand then immediately used to dilute a high concentration silkfibroin-based fragment solution (>4%) to the desired concentrations. Theresulting serums were more homogeneous and had a desirable smooth, clearlook and feel. Upon application to the skin, a white residue brieflyappeared that could be rubbed in. In an alternate method formulationswere created by dissolving HA in water and allowing it to sit for 1 dayuntil complete dissolution was observed. The HA and water solution wasthen used to dilute a high concentration silk fibroin-based fragmentsolution to the desired concentrations. The resulting serum was clear,smooth, homogeneous and left little to no white residue when applied.

Example 14 UV Hydrating Serums of the Present Disclosure

Variables tested include: concentration of HA, concentration of zincoxide, concentration of titanium dioxide, addition of vitamin C, andserum preparation method.

FIGS. 94A-94C are tables summarizing embodiments of cosmetic serums ofthe present disclosure with varying additives and concentrations ofcomponents suitable for protection against ultraviolet radiation (UV).Table 33 provides an embodiment of a hydrating serum of the presentdisclosure with vitamin C.

TABLE 33 Embodiment of Hydrating serum of the present disclosure withvitamin C % Silk Solution (60 minute boil, 25 kDA) 1.0% w/v HyaluronicAcid (sodium hyaluronate) 0.75% w/v Lemongrass Oil 20 uL/15 mL silksolution Sodium Ascorbyl Phosphate 6 g Lactic Acid 1.2 mL

A serum of the present disclosure can be made with from about 0.25% toabout 10% sodium hyaluronate (increasing % results in more viscousserum). 0.5% to about 10% silk solutions can be used to prepare a serumof the present disclosure. A serum of the present disclosure can beclear and have a yellow tinted color. A serum of the present discuslourecan have a pH=6. A serum of the present disclosure can have a lubricioustexture that is rubbed in easily without residue.

Concentration of HA:

Hyaluronic acid (Sodium Hyaluronate) was tested as an ingredient in theUV silk serum due to its hygroscopic properties and widely accepted usein cosmetic products to promote hydration of skin. 1%, 2.5% and 5% HAsolutions were tested. With increasing HA %, the serum became moreviscous and gel like. 1% HA was not feasible for the UV serum due to thefact that the UV additives (zinc oxide, titanium dioxide) are not watersoluble and need to be dispersed. 1% HA was not viscous enough fordispersion and the UV additives precipitated out. 2.5% gave the bestconsistency based on preferred feel, texture and viscosity and was ableto disperse the UV additives. 5% was a very thick, viscous serum.

Concentration of Mineral Filters: Zinc Oxide and Titanium Dioxide:

Zinc oxide and titanium dioxide were explored as UV additives that areconsidered safe. These additives mechanically protect from UV radiationby forming a physical reflective barrier on the skin. Both are notsoluble in water and must be dispersed for the current aqueous solution.Zinc oxide concentration varied from 2.5%, 3.75%, 5%, 5.625%, 10%, 12%and 15%. Titanium dioxide concentrations varied from 1.25%, 1.875%, 3%,5% and 10%. Increasing the concentration of UV additives resulted inminor increases of white residue and how well dispersed the additiveswere, however if mixed well enough the effects were negligible. Zincoxide and titanium dioxide were mixed together into serums in order toachieve broad spectrum protection. Zinc oxide is a broad spectrum UVadditive capable of protecting against long and short UV A and UV Brays. However titanium dioxide is better at UV B protection and oftenadded with zinc oxides for best broad spectrum protection. Combinationsincluded 3.75%/1.25% ZnO/TiO2, 5.625%/1.875% ZnO/TiO2, 12%/3% ZnO/TiO2,15%/5% ZnO/TiO2. The 3.75%/1.25% ZnO/TiO2 resulted in spf 5 and the5.625%/1.875% ZnO/TiO2 produced spf 8.

Vitamin C:

Sodium ascorbyl phosphate was used as a vitamin C source. Formulationswere created with the vitamin C concentration equal to that in the silkgel (0.67%). Formulations were also created with 20% sodium ascorbylphosphate which is soluble in water.

Serum Preparation:

The vitamin C (sodium ascorbyl phosphate) must first be dissolved inwater. Sodium hyaluronate is then added to the water, mixed vigorouslyand left to fully dissolve. The result is a viscous liquid (depending onHA %). The viscosity of the HA solution allows even dispersion of thezinc oxide and titanium dioxide and therefore HA must be mixed beforeaddition of UV additives. The zinc oxide and titanium dioxide are thenadded to the solution and mixed vigorously with the use of an electricblender. Silk solution is then added and mixed to complete the serumformulation.

Chemical Filters:

A UV serum of the present disclosure can include one, or a combinationof two or more, of these active chemical filter ingredients: oxybenzone,avobenzone, octisalate, octocrylene, homosalate and octinoxate. A UVserum of the present disclosure can also include a combination of zincoxide with chemical filters.

In an embodiment, a UV serum of the present disclosure can be appliedapproximately 15 minutes before sun exposure to all skin exposed to sun,and can be reapplied at least every 2 hours. In an embodiment, a UVserum of the present disclosure includes water, zinc oxide, sodiumhyaluronate, titanium dioxide, silk, and vitamin C or a vitamin Cderivative such as sodium ascorbyl phosphate. In an embodiment, a UVserum of the present disclosure protects skin and seals in moisture withthe power of silk protein. In an embodiment, a UV serum of the presentdisclosure improves skin tone, promotes collagen production anddiminishes the appearance of wrinkles and fine lines with theantioxidant abilities of vitamin C. In an embodiment, a UV serum of thepresent disclosure delivers moisture for immediate and long-termhydration throughout the day with concentrated hyaluronic acid. In anembodiment, a UV serum of the present disclosure helps prevent sunburnwith the combined action of zinc oxide and titanium dioxide. In anembodiment, a UV serum of the present disclosure is designed to protect,hydrate, and diminish fine lines while shielding skin from harsh UVA andUVB rays. In an embodiment, the silk protein in a UV serum of thepresent disclosure stabilizes and protects skin while sealing inmoisture, without the use of harsh chemical preservatives or syntheticadditives. In an embodiment, the vitamin C/derivative in a UV serum ofthe present disclosure acts as a powerful antioxidant that supports skinrejuvenation. In an embodiment, the sodium hyaluronate in a UV serum ofthe present disclosure nourishes the skin and delivers moisture forlong-lasting hydration. In an embodiment, the zinc oxide and titaniumdioxide in a UV serum of the present disclosure shields skin fromharmful UVA and UVB rays. The silk protein stabilization matrix in a UVserum of the present disclosure protects the active ingredients from theair, to deliver their full benefits without the use of harsh chemicalsor preservatives. The silk matrix also traps moisture within the skinfurthering the hydrating effect of the sodium hyaluronate.

Example 15 Dark Spot Films of the Present Disclosure

To reduce the appearance of dark spots, a high concentration of vitaminC may be necessary to reverse the overproduction of melanin. In thisexample, a 40% vitamin C (1.5:1 silk to vitamin C) was studied. The sizeand shape of the film can be made appropriate to a targeted area, forexample to a small circular film of diameter 1 in (2.54 cm).

The dark spot film, or a similar film of the present disclosure, ofvarying vitamin C concentration (0-50%) can be applied as a hydrofilm.Skin can be wetted with water. The film is then applied to the wet area.Water is then applied to the top surface of the film to turn it into agel. The gel can then be spread and gently massaged into the applicationarea. Table 34 provides details of an embodiment of a hydrofilm of thepresent disclosure (with no insoluble border).

TABLE 34 An Embodiment of a hydrofilm of the present disclosure % SilkSolution (60 minute boil, 2.56% 25 kDA) Quantity Vitamin C (1-ascorbic15.62 mg total (10 mg in 1 in acid) circle punch out) Volume of solutionper mold 2.44 mL Film Size 1.25 in diameter circle (7.917 cm{circumflexover ( )}2)

A film of the present disclosure can be made with different combinationsof % silk and volume to produce films with silk quantities of 3 mg/cm̂2to 10 mg/cm̂2. A film of the present disclosure can be made with fromabout 1% to about 50% 1-ascorbic acid. A film of the present disclosureis soluble in water (insoluble border is removed by punching out thecenter of the film). A film of the present disclosure can adhere to skinwith water. A film of the present disclosure can be spread on skin oncewater is applied. A film of the present disclosure can be dried when thehumidity of drying equipment is 16-40% and below the humidity of thelab. A film of the present disclosure can be clearr/transparent.

In an embodiment, a dark spot film of the present disclosure includeswater, silk, and vitamin C (L-ascorbic acid). In an embodiment, a darkspot film of the present disclosure includes 40% vitamin C. In anembodiment, a dark spot film of the present disclosure reduces skinpigmentation and evens skin tone in a targeted area with daily use.Vitamin C can inhibit pigment transfer from pigment producing cells,called melanocytes, to skin surface cells with continual application. Inan embodiment, a dark spot film of the present disclosure can be appliedto clean, dampened skin for 20 minutes. In an embodiment, additionalwater can be applied to an adhered film. The silk protein stabilizationmatrix in a dark spot film of the present disclosure protects the activeingredients from the air, to deliver their full benefits without the useof harsh chemicals or preservatives, such as paraben and phthalate.Thus, a dark spot film of the present disclosure is paraben andphthalate-free. Table 35 provides details of an embodiment of a film ofthe present disclosure.

TABLE 35 An Embodiment of a Film of the Present Disclosure % SilkSolution (60 minute boil, 25 kDA) 2.2% Surface area 5.07 cm{circumflexover ( )}2 Volume of silk solution for casting 1.56 mL Mass of silk perfilm: 34 mg Mass of 1-ascorbic acid per film: 23 mg Concentration of1-ascorbic acid in film:  40% pH 3

A 2.1% silk solution of the present disclosure (0.321 mL/cm̂2) to 2.4%silk solution of the present disclosure (0.282 mL/cm̂2) can been used tocreate dark spot films of the present disclosure with 34 mg of silk (6.7mg/cm̂2). In an embodiment, a 2.2% silk solution of the presentdisclosure (60 minute boil, 25 kDA) is used to produce a film of thepresent disclosure. The % silk and volume of solution can vary toproduce equivalent films. A dark spot film of the present disclosure canbe made with different combinations of % silk and volume to producefilms with silk quantities of 3 mg/cm̂2 to 10 mg/cm̂2. A dark spot film ofthe present disclosure can be made with from about 15 to about 50%1-ascorbic acid. A dark spot film of the present disclosure is solublein water (insoluble border). A dark spot film of the present disclosureis clear/transparent. A dark spot film of the present disclosure has apH=3 when water is applied. A dark spot film of the present disclosurecan adhere to skin with water. A dark spot film of the presentdisclosure can dry when humidity of drying equipment is 16-40% and belowthe humidity of the lab

Example 16 High Concentration Vitamin C Gels of the Present Disclosure

High concentration vitamin C gels were pursued up to 20%. Vitamin Ctype, vitamin C concentration, % silk and pH were varied to increase thequantity of vitamin C in a gel.

FIGS. 95A-95C are tables summarizing embodiments of high concentrationvitamin C gels of the present disclosure. The highest concentration ofvitamin C to gel was a 15% ascorbic acid 2 glucoside gel with 3.8% silksolution after 12 days. 5 and 10% ascorbic acid-2-glucoside formulationswith 2, 3 and 3.8% silk all gelled. For each group of % vitamin C,gelation first occurred in the 3.8% silk followed by the 3% and lastlythe 2%. It appears that there is a relationship between vitamin Cconcentration, silk concentration and gelation. If a solution has toomuch vitamin C in relation to silk, gelation will be prevented.Therefore, in order to produce high concentration vitamin C gels, higherconcentration silk is necessary. One sample was cast at 5.5% silk and20% vitamin C but gelation did not occur and a higher % silk may benecessary. Samples were also brought to a pH of 2 with lactic acid inorder to help induce gelation in 3% silk solutions with 10 or 20%vitamin C, however gelation did not occur in 12 days.

Example 17 Microbiological Study of Gels of the Present Disclosure

Contaminating micro-organisms in cosmetics may cause a spoilage of theproduct and, when pathogenic, they represent a serious health risk forconsumers worldwide. The United States Pharmacopoeia (USP) MicrobialLimits Test provides several methods for the determination of totalmicrobial count for bacteria, yeast and mold. Various gels of thepresent disclosure were tested to evaluate the possible microbialcontamination in three different states of their use (intact, in-use,ending product). FIG. 96 is a table summarizing the results of suchtesting.

The samples of gel and water samples from carboys were analyzed fordetermination of CFU/mL (colony-forming units per milliliter) of aerobicbacteria as well as yeast and mold. Samples were exposed to growthmedium of Tryptic Soy Agar (TSA) for bacteria and Potato Dextrose Agar(PDA) for fungi (yeast/mold) at an exposure temperature of 23±3° C.Samples were incubated at 30.0±2° C. for 3 days (bacteria) and 5 days(Fungi). Samples were then observed for determination of colony-formingunits/mL.

The limit of detection for the assays was 10 CFU/ml or g for bacteriaand fungi, and the values of <10 indicate that microorganisms could notbe detected in the samples. Values of >1.00E+04 indicate that themicrobial colonies are Too Numerous to Count in the dilutions plated.

Example 18 UV Silk Foams and Liquids of the Present Disclosure

In an embodiment, the vitamin C derivative sodium ascorbyl phosphate(DSM) was dissolved in water. Sodium hyaluronate (“HA”) was then addedto the water, mixed vigorously, and left to fully dissolve. The resultis a viscous liquid (depending on HA %). The viscosity of the HAsolution allows even dispersion of the zinc oxide and titanium dioxideand therefore HA is typically mixed before addition of UV additives. Thezinc oxide and titanium dioxide are added to the HA solution and mixedvigorously, for example with the use of an electric blender. 60 minuteboiled (˜25 kDa) silk solution is then added and mixed to create a 1%silk formulation.

Two formulations were created without the addition of sodium ascorbylphosphate (samples “HU2” and “HU4”). For sample HU2, zinc oxide andtitanium dioxide were added and mixed by blending with an electricblender and whisk. The result was a viscous white liquid (FIG. 98 andFIG. 99). Silk was then added and blended with an electric blender andwhisk. The solution became a creamy foam similar to shaving cream (FIG.97 and FIG. 100). Vitamin E in the form of dl-alpha tocopheryl acetatecan be added to the solution to recover a viscous liquid texture thatcan be applied with a smooth even texture (FIG. 98). With increasing thequantity of dl-alpha tocopheryl acetate, the formulation will becomeless foam-like and more of a smooth liquid or lotion texture.

HU4 was split into two batches: FIG. 99, batch 2 and FIG. 100, batch 1.The first batch followed the same procedures to HU2 and became a foam.For the second batch of HU4, sodium ascorbyl phosphate was added anddissolved before adding any zinc, titanium or silk. The UV additiveswere then added by blending with an electric blender and whisk andcreated a standard white viscous liquid. Silk was then added with anelectric blender and whisk. The result was slightly thicker viscousliquid than normally seen. Without wishing to be bound by theory, itappears the addition of sodium ascorbyl phosphate inhibits foaming.Without wishing to be bound by theory, it appears that whisking, asopposed to mixing or blending, creates a silk foam.

TABLE 36 Embodiments of UV Silk Foams and Liquids of the PresentDisclosure % HA Mass Sodium Total % (sodium Mass ZnO Mass AscorbylSample Volume silk hyaluronate) HA (g) % ZnO (g) % TiO₂ TiO₂ (g)Phosphate (g) HU2 55 1 2.5 1.375 12 6.6 3 1.65 N/A HU4 27.5 1 3.5 0.962512 3.3 3 0.825 5.5 Batch 1 HU4 27.5 1 3.5 0.9625 12 3.3 3 0.825 N/ABatch 2

Example 19 Lyophilized Silk Powders of the Present Disclosure

TABLE 37 Embodiments of lyophilized silk powders Silk Solution TreatmentSoluble ~60 kDa silk, 6% silk, lyopholize and cut with no pH = 7-8blender ~60 kDa silk, 6% silk, lyopholize and cut with no pH = 10blender ~25 kDa silk, 6% silk, lyopholize and cut with yes pH = 7-8blender ~25 kDa silk, 6% silk, lyopholize and cut with yes pH = 10blender

The above silk solutions were transformed to a silk powder throughlyophilization to remove bulk water and chopping to small pieces with ablender. pH was adjusted with sodium hydroxide. Low molecular weightsilk (˜25 kDa) was soluble while high molecular weight silk (˜60 kDa)was not.

The lyophilized silk powder can be advantageous for enhanced storagecontrol ranging from 10 days to 10 years depending on storage andshipment conditions. The lyophilized silk powder can also be used as araw ingredient in the pharmaceutical, medical, consumer, and electronicmarkets. Additionally, lyophilized silk powder can be re-suspended inwater, HFIP, or an organic solution following storage to create silksolutions of varying concentrations, including higher concentrationsolutions than those produced initially.

In an embodiment, aqueous pure silk fibroin-based protein fragmentsolutions of the present disclosure comprising 1%, 3%, and 5% silk byweight were each dispensed into a 1.8 L Lyoguard trays, respectively.All 3 trays were placed in a 12 ft² lyophilizer and a single runperformed. The product was frozen with a shelf temperature of ≦−40° C.and held for 2 hours. The compositions were then lyophilized at a shelftemperature of −20° C., with a 3 hour ramp and held for 20 hours, andsubsequently dried at a temperature of 30° C., with a 5 hour ramp andheld for about 34 hours. Trays were removed and stored at ambientconditions until further processing. Each of the resultant lyophilizedsilk fragment compositions were able to dissolve in aqueous solvent andorganic solvent to reconstitute silk fragment solutions between 0.1 wt %and 8 wt %. Heating and mixing were not required but were used toaccelerate the dissolving rate. All solutions were shelf-stable atambient conditions.

In an embodiment, an aqueous pure silk fibroin-based protein fragmentsolution of the present disclosure, fabricated using a method of thepresent disclosure with a 30 minute boil, has a molecular weight ofabout 57 kDa, a polydispersity of about 1.6, inorganic and organicresiduals of less than 500 ppm, and a light amber color.

In an embodiment, an aqueous pure silk fibroin-based protein fragmentsolution of the present disclosure, fabricated using a method of thepresent disclosure with a 60 minute boil, has a molecular weight ofabout 25 kDa, a polydispersity of about 2.4, inorganic and organicresiduals of less than 500 ppm, and a light amber color.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 6 kDa to about 16 kDa includes the steps of:degumming a silk source by adding the silk source to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 60° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in an ovenhaving a temperature of about 140° C. for a period of at least 1 hour;removing the lithium bromide from the silk fibroin extract; andproducing an aqueous solution of silk protein fragments, the aqueoussolution comprising: fragments having an average weight averagemolecular weight ranging from about 6 kDa to about 16 kDa, and whereinthe aqueous solution of pure silk fibroin-based protein fragmentscomprises a polydispersity of between about 1.5 and about 3.0. Themethod may further comprise drying the silk fibroin extract prior to thedissolving step. The aqueous solution of pure silk fibroin-based proteinfragments may comprise lithium bromide residuals of less than 300 ppm asmeasured using a high-performance liquid chromatography lithium bromideassay. The aqueous solution of pure silk fibroin-based protein fragmentsmay comprise sodium carbonate residuals of less than 100 ppm as measuredusing a high-performance liquid chromatography sodium carbonate assay.The method may further comprise adding a therapeutic agent to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a molecule selected from one of anantioxidant or an enzyme to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga vitamin to the aqueous solution of pure silk fibroin-based proteinfragments. The vitamin may be vitamin C or a derivative thereof. Theaqueous solution of pure silk fibroin-based protein fragments may belyophilized. The method may further comprise adding an alpha hydroxyacid to the aqueous solution of pure silk fibroin-based proteinfragments. The alpha hydroxy acid may be selected from the groupconsisting of glycolic acid, lactic acid, tartaric acid and citric acid.The method may further comprise adding hyaluronic acid or its salt format a concentration of about 0.5% to about 10.0% to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding at least one of zinc oxide or titanium dioxide. A filmmay be fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The film may comprise fromabout 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivativethereof. The film may have a water content ranging from about 2.0 wt. %to about 20.0 wt. %. The film may comprise from about 30.0 wt. % toabout 99.5 wt. % of pure silk fibroin-based protein fragments. A gel maybe fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The gel may comprise fromabout 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivativethereof. The gel may have a silk content of at least 2% and a vitamincontent of at least 20%.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 17 kDa to about 38 kDa includes the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of between about 30 minutes to about 60minutes so as to result in degumming; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 80° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in a dry ovenhaving a temperature in the range between about 60° C. to about 100° C.for a period of at least 1 hour; removing the lithium bromide from thesilk fibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, wherein the aqueous solutionof silk protein fragments comprises sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, wherein the aqueous solution ofpure silk fibroin-based protein fragments comprises fragments having anaverage weight average molecular weight ranging from about 17 kDa toabout 38 kDa, and wherein the aqueous solution of pure silkfibroin-based protein fragments comprises a polydispersity of betweenabout 1.5 and about 3.0. The method may further comprise drying the silkfibroin extract prior to the dissolving step. The aqueous solution ofpure silk fibroin-based protein fragments may comprise lithium bromideresiduals of less than 300 ppm as measured using a high-performanceliquid chromatography lithium bromide assay. The aqueous solution ofpure silk fibroin-based protein fragments may comprise sodium carbonateresiduals of less than 100 ppm as measured using a high-performanceliquid chromatography sodium carbonate assay. The method may furthercomprise adding a therapeutic agent to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga molecule selected from one of an antioxidant or an enzyme to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a vitamin to the aqueous solution ofpure silk fibroin-based protein fragments. The vitamin may be vitamin Cor a derivative thereof. The aqueous solution of pure silk fibroin-basedprotein fragments may be lyophilized. The method may further compriseadding an alpha hydroxy acid to the aqueous solution of pure silkfibroin-based protein fragments. The alpha hydroxy acid may be selectedfrom the group consisting of glycolic acid, lactic acid, tartaric acidand citric acid. The method may further comprise adding hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding at least one of zinc oxide ortitanium dioxide. A film may be fabricated from the aqueous solution ofpure silk fibroin-based protein fragments produced by this method. Thefilm may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin Cor a derivative thereof. The film may have a water content ranging fromabout 2.0 wt. % to about 20.0 wt. %. The film may comprise from about30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based proteinfragments. A gel may be fabricated from the aqueous solution of puresilk fibroin-based protein fragments produced by this method. The gelmay comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or aderivative thereof. The gel may have a silk content of at least 2% and avitamin content of at least 20%.

According to aspects illustrated herein, there is disclosed a method forpreparing an aqueous solution of pure silk fibroin-based proteinfragments having an average weight average molecular weight ranging fromabout 39 kDa to about 80 kDa, the method including the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of about 30 minutes so as to result indegumming; removing sericin from the solution to produce a silk fibroinextract comprising non-detectable levels of sericin; draining thesolution from the silk fibroin extract; dissolving the silk fibroinextract in a solution of lithium bromide having a starting temperatureupon placement of the silk fibroin extract in the lithium bromidesolution that ranges from about 80° C. to about 140° C.; maintaining thesolution of silk fibroin-lithium bromide in a dry oven having atemperature in the range between about 60° C. to about 100° C. for aperiod of at least 1 hour; removing the lithium bromide from the silkfibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, fragments having an averageweight average molecular weight ranging from about 40 kDa to about 65kDa, and wherein the aqueous solution of pure silk fibroin-based proteinfragments comprises a polydispersity of between about 1.5 and about 3.0.The method may further comprise drying the silk fibroin extract prior tothe dissolving step. The aqueous solution of pure silk fibroin-basedprotein fragments may comprise lithium bromide residuals of less than300 ppm as measured using a high-performance liquid chromatographylithium bromide assay. The aqueous solution of pure silk fibroin-basedprotein fragments may comprise sodium carbonate residuals of less than100 ppm as measured using a high-performance liquid chromatographysodium carbonate assay. The method may further comprise adding atherapeutic agent to the aqueous solution of pure silk fibroin-basedprotein fragments. The method may further comprise adding a moleculeselected from one of an antioxidant or an enzyme to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding a vitamin to the aqueous solution of pure silkfibroin-based protein fragments. The vitamin may be vitamin C or aderivative thereof. The aqueous solution of pure silk fibroin-basedprotein fragments may be lyophilized. The method may further compriseadding an alpha hydroxy acid to the aqueous solution of pure silkfibroin-based protein fragments. The alpha hydroxy acid may be selectedfrom the group consisting of glycolic acid, lactic acid, tartaric acidand citric acid. The method may further comprise adding hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding at least one of zinc oxide ortitanium dioxide. A film may be fabricated from the aqueous solution ofpure silk fibroin-based protein fragments produced by this method. Thefilm may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin Cor a derivative thereof. The film may have a water content ranging fromabout 2.0 wt. % to about 20.0 wt. %. The film may comprise from about30.0 wt. % to about 99.5 wt. % of pure silk fibroin-based proteinfragments. A gel may be fabricated from the aqueous solution of puresilk fibroin-based protein fragments produced by this method. The gelmay comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or aderivative thereof. The gel may have a silk content of at least 2% and avitamin content of at least 20%.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While themethods of the present disclosure have been described in connection withthe specific embodiments thereof, it will be understood that it iscapable of further modification. Further, this application is intendedto cover any variations, uses, or adaptations of the methods of thepresent disclosure, including such departures from the presentdisclosure as come within known or customary practice in the art towhich the methods of the present disclosure pertain.

1. A composition comprising: pure silk fibroin-based protein fragmentsthat are substantially devoid of sericin, wherein the composition has anaverage weight average molecular weight ranging from about 17 kDa toabout 38 kDa, wherein the composition has a polydispersity of betweenabout 1.5 and about 3.0, wherein the composition is substantiallyhomogeneous, wherein the composition includes between 0 ppm to about 500ppm of inorganic residuals, and wherein the composition includes between0 ppm to about 500 ppm of organic residuals.
 2. The composition of claim1 wherein the inorganic residuals include lithium bromide and whereinthe organic residuals include sodium carbonate.
 3. The composition ofclaim 2 wherein the lithium bromide residuals are measurable using ahigh-performance liquid chromatography lithium bromide assay, and thesodium carbonate residuals are measurable using a high-performanceliquid chromatography sodium carbonate assay.
 4. The composition ofclaim 1 further comprising less than 10% water.
 5. The composition ofclaim 1 wherein the pure silk fibroin-based protein fragments are fromabout 0.1 wt % to about 30.0 wt %.
 6. The composition of claim 1 in theform of a lyophilized form or structure.
 7. The composition of claim 1in the form of a solution.
 8. The composition of claim 7 wherein thepure silk fibroin-based protein fragments are stable in the solution. 9.The composition of claim 7 wherein the solution is an aqueous solution.10. The composition of claim 7 wherein the solution is an organicsolution.
 11. The composition of claim 1 in a sealed container.
 12. Thecomposition of claim 1 further comprising one or more molecules selectedfrom the group consisting of therapeutic agents, growth factors,antioxidants, proteins, vitamins, carbohydrates, polymers, nucleicacids, salts, acids, bases, biomolecules, glycosamino glycans,polysaccharides, extracellular matrix molecules, metals, metal ion,metal oxide, synthetic molecules, polyanhydrides, cells, fatty acids,fragrance, minerals, plants, plant extracts, preservatives and essentialoils.
 13. The composition of claim 12 in the form of a solution.
 14. Thecomposition of claim 13 wherein the pure silk fibroin-based proteinfragments are stable in the solution.
 15. The composition of claim 12wherein the vitamin is vitamin C or a derivative thereof.
 16. Thecomposition of claim 12 further comprising an alpha hydroxy acidselected from the group consisting of glycolic acid, lactic acid,tartaric acid and citric acid.
 17. The composition of claim 12 furthercomprising hyaluronic acid or its salt form at a concentration of about0.5% to about 10.0%.
 18. The composition of claim 12 further comprisingat least one of zinc oxide or titanium dioxide.
 19. The composition ofclaim 1 wherein the pure silk fibroin-based protein fragments arehypoallergenic.
 20. The composition of claim 1 having less than 10colony forming units per gram.